Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND PRODUCTS FOR PEPTIDE-BASED
DNA SEQUENCE CHARACTERIZATION AND ANALYSIS
FIELD OF INVENTION
This invention relates to the fields of Molecular
Biology and Genetics, with particular reference to the
identification and analysis of DNA molecules.
BACKGROUND
In biology and medicine, there is frequently a
need to determine the sequence of a DNA fragment. The
to fragment may be derived from genomic DNA of viral,
procaryotic or eucaryotic origin, or it may be a derived
from cDNA. In many-cases, the fragment derives from a
larger DNA molecule, or set of molecules, whose sequence
(here defined as the reference sequence) is already known.
Such cases are not rare and will become increasingly common
as more and more natural DNA and cDNA sequences are
deposited in available databases.
A number of methods presently exist fox
determining the nucleotide sequence of a DNA fragment. The
most commonly applied method involves cloning the fragment
in a plasmid vector of known sequence, purifying the
plasmid DNA, annealing a primer complimentary to a portion
of the known sequence to one strand of the molecule,
extending the primer with DNA polymerase, terminating the
polymerization with dideoxy nucleotides, and comparing the
lengths of the various terminated molecules to reveal the
nucleotide sequence 3' to the primer. Other DNA sequencing
methods exist, such as selective cleavage or sequencing by
hybridization to biochips. All of these methods are based
solely on in vitro DNA chemistry and biochemistry. Other
well developed methods, such as SSCP (single strand
conformational polymorphism analysis), heteroduplex
sensitivity to nuclease analysis (EMD), and allele-specific
oligonucleotide hybridization, (ASO) exist for detecting
mutations or sequence polymorphisms in DNA fragments.
These methods, too, are based solely on in vitro DNA
chemistry and biochemistry.
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Rather than examining a DNA molecule by analyzing
the DNA itself, in the invention described here the DNA is
incorporated into a hybrid artificial gene that is
transcribed and translated to produce a hybrid peptide.
Physical analysis of the peptide, in conjunction with
informatic analysis of the reference sequence, allows one
to identify the sequence of the DNA molecule.
The analysis of peptide size as a means to infer
information about a gene goes back to at least 1965, when
it was reported that phage T4 amber mutants made truncated
proteins and that the size of the peptide made in an amber
mutant was approximately proportional to the distance of
the mutation from the 3' end of the gene. In recent years,
this phenomenon has provided the basis for the protein
truncation assay for identifying nonsense and frameshift
mutations in mammalian genes. In the protein truncation
assay, an exon is assayed for chain termination mutations
by PCR-amplifying the exon, expressing it in a cell free
transcription/translation system, and examining the
expressed polypeptide by SDS polyacrylamide gel
electrophoresis to determine if it is smaller than a
non-mutant control polypeptide. While the protein
truncation assay can reveal the presence of a nonsense or
frameshift mutation, it is important to note that the assay
does not reveal the molecular nature or exact location of
the mutation - one does not know if it is a TAG, TGA, TAA
or frameshift mutation, and one only knows the approximate
location of the mutation within the exon.
There presently exists well developed art by
which "unknown" proteins are identified by means of coupled
physical and informatic analysis. In these cases, one
begins with a naturally occurring protein (or sometimes a
fusion protein containing a natural amino sequences) and
uses the coupled analysis to determine the protein's
identity - for example, by mass spectrometric analysis of
tryptic fragment masses followed by search of a database of
in silico-generated tryptic fragments, in which the
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sequences that are the sources of the tryptic fragment data
may be taken from existing protein sequence databases or
may be created by in silico translation of existing nucleic
acid databases. In other cases, mass analysis of peptides
derived from known proteins has been used to identify
sequence deviations from previously determined .protein
sequences.
Whereas the database search activities in the
prior art (examples of which are referred to above) are
aimed at protein identification and/or analysis, in the
instant invention the search activity is aimed at DNA
identification or analysis. Thus the two are distinctly
different in concept and practice. The artificial hybrid
peptides that are analyzed in the instant invention are not
naturally occurring, nor are they necessarily biologically
active. And yet they have distinct utility as reporters
that carry information about the nucleic acids that encode
them.
The analysis of peptide reporters provides a
number of clear advantages over analysis of the DNA
sequences that encode them. One advantage derives from the
fact that a peptide is considerably smaller than the DNA
that encodes it (individual amino acids averages about 110
Da each whereas the trinucleotides (triplets) that encode
them average over N Daltons each). Another advantage
derives from the fact that peptides are much more diverse
in composition than nucleic acids, as they are composed of
combinations of 20 different amino acids instead of
combinations of 4 different nucleotides. Thus, by way of
illustration, two random DNA fragments of identical
composition (e.g., with 10 adenines, 10 thymines, 15
guanines, and 15 cytosines) are extremely unlikely to
encode peptides of identical composition, and so, whereas
the two nucleic acids have identical masses and cannot be
distinguished on the basis of mass, the peptides that they
encode will, except in statistically very rare cases, have
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different masses and can be readily distinguished on the
basis of mass.
SUMMARY OF THE INVENTION
In the invention described here the DNA to be
analyzed is incorporated into a hybrid artificial gene that
is then transcribed and translated to produce a hybrid
peptide. Analysis of the peptide, rather than analysis of
the DNA, is used to gain sequence data about the DNA.
Specifically, the mass and/or composition and/or
partial or complete amino acid sequence of the hybrid
peptide is determined, and the data are used to search for
matches in data sets produced by in silico transcription
and translation of hybrid artificial genes created in
silico using the reference sequence, or using
transformations of the reference sequence such as single
nucleotide deletions or substitutions thereof. This
peptide-based approach to DNA sequence-determination is
fundamentally different from all other methods in the art,
none of which employs transcription, translation and
peptide analysis, as does the instant invention.
It is important to emphasize that the peptides
that are produced and analyzed in the course of practicing
the invention are not derived from naturally occurring
proteins, nor did they exist anywhere prior to their
production from the hybrid artificial genes. Likewise the
hybrid artificial genes of the invention never existed in
nature prior to their production in the course of
practicing the invention.
Expected properties of peptides translated from
unknown nucleotide sequences
The invention depends on means to translate a
portion of the unknown sequence as part of a fusion peptide
whose synthesis originates in the known sequence and
extends into the unknown sequence that is being
characterized. The unknown sequence need not comprise
actual protein-coding sequence in the cell from which it
originates, although it may in some cases, and so the
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invention is of general applicability and not confined to
coding sequences. The invention also depends on means to
accurately measure the mass and/or composition and/or
partial or complete amino acid sequence of the fusion
peptide. Many methods for making such measurements are
known in the art, and a number of them will be discussed
later in this specification. But first, let us consider
the issue of the expected sizes, masses, and amino acid
sequences of the peptides that can be translated from an
unknown sequence. For the purpose of this analysis, we
will make the simplifying assumption that the unknown
sequence is statistically random. Later in this
specification, specific examples using natural DNA
sequences will be provided.
Of the 64 colons, 3 (UAA, UAG, UGA) are nonsense
colons that terminate translation. Thus, in any reading
frame of a random nucleotide sequence, approximately 1 of
21 colons (-3/64) will be nonsense and approximately 20 of
21 (61/64) will be sense colons.
We now ask the question: if translation begins at
an arbitrary nucleotide in a random DNA sequence, how large
will the resulting peptide be? The answer can be given in
the form of a distribution that can be calculated as
follows. The likelihood that the first colon in the
sequence is a nonsense colon (and that the peptide will
thus be zero amino acids in length) is 1/21, or -4.7%. The
likelihood that the first colon is not a nonsense colon and
the second colon is a nonsense colon .(and that the peptide
will thus be one amino acid in length) is 20/21 x 1/21, or
-4.5%. The likelihood that the first and second colons are
not nonsense colons and the third colon is a nonsense colon
(and that the peptide will thus be two amino acids in
length) is 20/21 x 20/21 x 1/21, or ~4.3%, and so on. Thus
the likelihood that a peptide will have exactly length N is
given by the expression (20/21)N x 1/21. Also, since the
chance that a peptide will reach at least length N is
(20/21)N, we can readily calculate the likelihood of a
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peptide having a given length or less from the expression
1- (20/21)N.
The table below shows the calculated
probabilities, for the first 24 codons of a random DNA
sequence, that a given peptide will be of a given length or
less. The table indicates that, for example, 0.705
(approximately 70%) of all peptides will be 24 or fewer
amino acids in length, and that 0.216 (approximately 20%)
of all peptides will be 4 or fewer amino acids in length.
In other words, about half of all peptides will be between
5 and 24 amino acids in length.
Per cent of length N
Peptide length (N) or less (1- (20/21)N)
0 4.7
1 9.3
2 13.6
3 17.7
4 21.6
5 25.4
6 28.9
7 32.3
8 35.5
9 38.6
10 41.5
11 44.3
12 47.0
13 49.5
14 51.9
15 54.2
16 56.4
17 58.4
18 60.0
19 62.3
20 64.1
21 65.8
22 67.4
23 69.0
24 70.5
These expectations were tested by taking the
10,942 base pair sequence that includes the entire human
nucleolin gene (Genbank accession number gb J05584) and
translating it in silico beginning at number of arbitrarily
chosen positions. In particular, translation was begun at
every 50th nucleotide beginning at position 2001 and ending
at position 4001. The lengths of the encoded peptides,
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translated from the indicated position to the first
in-frame nonsense codon encountered, are listed below.
Those between 5 and 24 amino acids are marked with an
asterisk. Seventeen out of the 40 peptides are between 4
and 24 amino acids in length, very close to the 20 out of
40 predicted on theoretical grounds, as described above.
Start Peptide length (amino acids)
2001 24*
2051 2
2101 21*
2151 20*
2201 45
2251 2
2301 20*
2351 37
2401 16*
2451 21*
2501 25
2551 30
2601 0
2651 20*
2701 13*
2751 11*
2801 0
2851 4*
2901 21*
2951 16*
3001 14*
3051 0
3101 69
3151 1
3201 26
3251 19*
3301 211
3351 107
3401 86
3451 161
3501 79
3551 36
3601 111
3651 7*
3701 42
3751 61
3801 0
3851 38
3901 11*
3951 40
4001 1g*
If the nucleotide sequence is random, the
probability that a sequence of a given length translated
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from it will have a particular amino acid sequence can be
calculated simply by multiplying together the frequencies
in the genetic code of the codons encoding each amino acid
amino acid in the sequence. Since some amino acids have as
many as six codons and others as few as one, the predicted
frequency will vary depending on the amino acid sequence
itself. Thus the sequence LRRLLR, made up entirely of
six-codon amino acids, will appear. at a frequency of 1 in
(6/61)6, or approximately once in one million codons, and
the sequence MWWMMW, made up entirely of one-codon amino
acids, will appear at a frequency of 1 in (1/61)6, or
approximately once. in fifty billion codons. The
frequencies of other sequences will fall between these two
extremes. The important point for us is that even a
relatively short sequence will appear very rarely, and so
if we can determine the amino acid sequence of a peptide
translated from unknown sequence, we can match it to a
portion of the reference sequence with high specificity.
Let us now address the issue of the degree of
specificity that can be obtained in a search of the
reference sequence if we know only the mass, but not the
amino acid sequence or composition, of a peptide that is
translated from an unknown portion of it? For the sake of
this discussion, we will assume that the mass of the
peptide is determined with such accuracy as to distinguish
each amino acid combination from all others. The number of
distinct amino acid combinations and their frequencies is
represented by the polynomial expansion
(a+b+c+d+......+q+r+s)N, where the letters "a" through "s"
(19 letters) represent the frequencies in the genetic code
of each amino acid (there are 19 instead of 20 letters
because two amino acids, leucine and isoleucine, have the
same mass and must be treated as a group) and N represents
the length of the peptide. The number of terms in the
expansion represents the number of composition classes, and
the value of each term divided by the sum of the values of
all of the terms gives the frequency of any given class.
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It should be clear to the reader that for all but very
small values of N, the frequency of any given class will be
very low.
Depending on the size and sequence of the
reference sequence, there may be just one peptide encoded
in it of a given mass, or there may be more than one.
Generation of fusion peptides
The operation of the invention depends upon the
presence of a specially engineered DNA sequence adjacent to
the unknown DNA. The engineered sequence contains at
minimum the following elements: (1) a promoter sequence
oriented to promote transcription into the unknown
sequence, and (2) a translation initiation sequence, and
(3) a coding sequence comprises at minimum a start codon.
Transcription from the promoter, followed by translation of
the transcript beginning at the start codon, yields a
fusion peptide with an N-terminal portion of known amino
acid composition followed by a portion of unknown sequence
encoded by the unknown DNA. A second known sequence may,
in some embodiments, be incorporated into the C-terminal
portion of the fusion peptide.
Analysis of fusion peptides
Once a fusion peptide has been produced as
described above, it must be analyzed to determine its mass
and/or its composition and/or its amino acid sequence.
(Mass Spectrometry is one preferred analytical method
because it is fast and highly accurate. A number of
specific examples of the application of mass spectrometric
analysis to fusion peptides are given later in this
specification. The data are compared with the data set
generated in silico that contains all possible fusion
peptides generated by fusing the known sequence to the
reference sequence at all possible positions in the
reference sequence and calculating the masses and/or
compositions and/or amino acid sequences of the resulting
peptides. Absence of a match, which will occur in the
great majority of the positions, allows one to exclude that
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portion of the reference sequence from consideration,
whereas a match indicates that it may indeed be the actual
sequence coding for the unknown portion of the fusion
peptide. If there is only one such match, and if the
entire reference sequence has been scanned, then the
unknown sequence has been identified. If there are
multiple matches, additional data are needed to narrow the
conclusion to a single site. Such data can come in a
number of forms, including the generation and analysis of
more than one fusion peptide from the same region the
reference sequence, or the generation and analysis of
peptides translated .from different reading frames of the
same nucleic acid sequence. Specific examples of multiple
peptide analysis from nearby, adjacent or overlapping
nucleotides are given below and in the claims. But it is
important to state that the invention has utility even if
it narrows down, but does not absolutely define, the
identity of the unknown sequence.
Purification of fusion proteins prior to analysis
In some cases it may be desirable to purify the
fusion peptide prior to analysis. One well established
means for doing this is to include a predetermined amino
acid sequence (epitope tag) in the known portion of the
fusion peptide that binds to a known molecule (e.g., an
antibody) or other reagent (immobilized nickel, for
example) . The antibody or other reagent is then used to
capture and purify the peptide by immunoaffinity
chromatography or immobilized metal affinity chromatography
(IMAC) prior to analysis. Or a larger known sequence
suitable for affinity purification such as
glutathione-S-transferase (GST), thioredoxin, or maltose
binding protein(MBP), may be incorporated at the N or
C-terminus of the peptide. Many means for separating
and/or purifying peptides or proteins are also well known
and may be applied in certain embodiments of the invention.
These include gel electropharesis, capillary
electrophoresis, liquid chromatography (LC), capillary
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liquid chromatography, high performance liquid
chromatography (HPLC), differential centrifugation,
filtration, gel filtration, membrane chromatography,
affinity purification, biomolecular interaction analysis
(BIA), ligand affinity purification,
glutathione-S-transferase affinity chromatography,
cellulose binding protein affinity chromatography, maltose
b i nd i ng protein of f inity chromatography,
avidin/streptavidin affinity chromatography, S-tag affinity
chromatography, thioredoxin affinity chromatography,
metal-chelate affinity chromatography, immobilized metal
affinity chromatography, epitope-tag affinity
chromatography, immunoaffinity chromatography,
immunoaffinity capture, capture using bioreactive mass
spectrometer probes, mass spectrometric immunoassay, and
immunoprecipitation.
Detection and characterization of mutations and DNA
polvmorphisms
Certain embodiments of the invention can be used
to detect and characterize naturally occurring mutations
and DNA polymorphisms, including single nucleotide
polymorphisms {SNPs). This is done by comparing the coding
capacity of subsets of the reference sequence with the
coding capacity of equivalent subsets of the sequence
derived from it by specific nucleotide changes, as follows.
(By coding capacity is meant the set of the amino acids
encoded in at least one reading frame of a sequence; a
change in the coding capacity would be-due, at minimum, to
a change in amino acid composition of at least one encoded
peptide.) For every peptide generated in silico by
translation of a sequence containing a portion of the
reference sequence as described previously in this
specification, an additional related set of peptides is
generated by generating, also in silico, a set of
transformed DNA sequences derived from the same portion of
the reference DNA sequence, each member of the set
containing a different sequence alteration. Each member of
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the transformed set is then translated in silico to give a
transformed set of peptide sequences. In the case of
single nucleotide substitutions, for example, since there
are exactly three nucleotide changes that can be made at
each position in the relevant portion of the reference DNA
sequence, the expanded set of peptides will contain 3N
members, where N is the length of the relevant portion of
the reference nucleotide sequence. (In most cases, some of
the members of the new set will be identical due to the
degeneracy of the genetic code.) When the transformed data
set is searched with the experimentally determined peptide
data, as described previously in this specification, single
nucleotide departures from the reference sequence are
revealed as matches to members of the transformed data set.
In another embodiment of the invention, mutations
or DNA polymorphisms are detected and quantified, by first
producing a PCR amplicon representing a distinct portion of
the reference sequence, such as a single exon in a gene of
interest . The amplicon is expressed as part of a fusion
peptide as described previously. In one embodiment, the
exon is expressed in frame with respect to the translation
initiation codon in the vector, with the result that the
peptide comprises the entire amino acid sequence encoded in
the exon. If the PCR template contains a point mutation
that alters the amino acid sequence, this will be observed
as, for example, a distinct change in the mass of the
peptide relative to the mass of the peptide from the
non-mutant exon. A large number of diseases are known to
be caused by mutations in known genes, and the mutations in
these genes that are responsible for dominant or recessive
genetic disease may be examined using the instant
invention. These include: Ataxia talangietasia (ATM),
Familiars edematous polypsosis (APC), Hereditary
breast/ovarian cancer (BRCAl, BRCA2), Hereditary melanoma
(CDK2, CDKN2), Hereditary non-polypsosis colon cancer
(hMSH2, hMLHl, hPMSl, hPMS2), Hereditary retinoblastoma
(RB1), Hereditary Wilm's Tumor (WT1), Li-Fraumeni syndrome
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(p53), Multiple endocrine neoplasia (MEN1, MEN2), Von
Hippel-Lindau syndrome (VHL), Congenital adrenal
hyperplasia, Androgen Receptor Mutation,
Tetrahydrobiopterin deficiency, X-Linked
agammaglobulinemia, Cystic Fibrosis (CFTR), Muscular
Dystrophy (DMD, BMD), Factor X deficiency, Mitochondrial
gene deficiency, Factor VII deficiency, Glucose-6-Phosphate
deficiency, Pompe Disease, Hemophilia A, Hexosaminidase A
deficiency, Human Type I and Type III Collagen deficiency
X-linked SCID, Retinitis pigmentosa (RP) LIACAM deficiency,
MCAD deficiency, LDL Receptor deficiency, Ornithine
Transcarbamylase deficiency, PAX6 Mutation,
Phenylketonuria, Tuberous Sclerosis, von Willebrand Factor
Disease, Werner Syndrome.
DESCRIPTION OF PREFERRED EMBODIMENTS
Specific Examples
In examples 1-6 to follow, the masses of the
peptides encoded in the various nucleotide sequences were
calculated using the table of mass values shown below
Peptide masses calculated using these values were rounded
off to the nearest Dalton.
Amino Acid Mass
Alanine 71.0 Da
Arginine 156.1
Asparagine 114.0
Aspartic acid 115.0
Cysteine 103.0
Glutamic acid 129.0
Glutamine 128.1
Glycine 57.p
Histidine 137.1
Isoleucine 113.1
Leucine 113.1
Lysine 128.1
Methionine 131.0
Phenylalanine 147.1
Proline 97.1
Serine g7.p
Threonine 101.0
Tryptophan 186.1
Tyrosine 163.1
Valine 99.1
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Example 1. Identification of a subcloned EcoRI fragment
of a cloned human crene .
The EMBL3 clone HG3 contains a 10942 base pair
insert containing the human nucleolin gene as well as
surrounding intergenic sequences (Srivistava, Genbank
accession number gb J05584). Purified HG3 DNA is digested
to completion with the restriction endonuclease EcoRI and
a plasmid mini-library is constructed by cloning the
fragments into the EcoRI site of the vector pUCl9 using
standard methods. The library is transformed into
competent E. coli BLR cells. Ampicillin resistant colonies
are selected on LB ampicillin plates, and a single colony
is picked and used to prepare a plasmid miniprep. A 250 ml
liquid culture of cells from this colony is grown in
LB-ampicillin medium at 25 degrees to a density of 2 x 108
cells per ml, induced with 1 mM IPTG for 2 hours,
concentrated to a volume of 10 ml by centrifugation, and
lysed by sonication in the presence of the protease
inhibitors AEBSF, bestatin, E-64 and pepstatin A. A second
250 ml control culture with nonrecombinant pUCl9 vector is
prepared in parallel. All of the above steps follow
standard methods well known in the art.
A 10 ul aliquot of each cell lysate is subjected
to capillary liquid chromatography (LC) followed by
electrospray ionization mass spectrometry (ESI/MS) using
methods and procedures well known in the art. The spectrum
of the lysate from the induced cells is observed to contain
a distinct peak, at a position corresponding to a mass of
5253 2 Daltons that is not observed in the control cell
lysate.
To identify the nucleotide sequence responsible
for the 5253 peak, the J05584 sequence is scanned to
identify each EcoRI site. Five such sites are identified.
Each EcoRI fragment is ligated, in silico, to the EcoRI
site in the pUCl9 vector, producing 10 possible recombinant
plasmids, one for each of the two possible orientations of
each insert in the vector. The predicted amino acid
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sequence and molecular mass of each IPTG-inducible hybrid
translation product (translated from the mRNA transcribed
from the lac promoter in the vector) is calculated, and the
masses of the ten possible polypeptides are tabulated, as
shown in the table below.
Position of EcoRI site Orientation in pUCl9 Predicted Peptide Mass
3190 forward 7070 Daltons
3190 reverse 5253
4028 forward 3998
4028 reverse 5268
6066 forward 4969
6066 reverse 2726
9241 forward 8485
9241 reverse 3109
9543 forward 2840
9543 reverse 3878
The mass values above were computed by
translating each hypothetical fusion polypeptide and
removing the N-terminal methionine.
Comparison of the experimental results with the
values in the table indicates reveals a match to the
predicted mass value for one of the ten candidates -
specifically the sequence that begins at position 3190 of
the reference sequence and proceeds from right to left.
Retrieval of the reference sequence beginning at position
3190 indicates that the cloned sequence begins with
"GAATTCTTACACCTCATACTTTCCCAAGCCCCA.ACTTTCTCATCTGAAAATGGTAA
TAGTATCATCCTTACATGTTTAAGGTCATGAATTGCTATGTGTA..." (first 100
nucleotides shown). The identification is confirmed by
dideoxy sequencing from a primer 150 .nucleotides upstream
of the junction between the pUCl9 sequence and the EcoRI
fragment.
In this example the starting material was a
cloned gene. If one begins instead with a cloned a cDNA
library and uses identical procedures in an iterative
manner, the identity of multiple members of the library are
ascertained.
Example 2. Identification of a subcloned EcoRI fragment
of a cloned human ctene usincr peptide affinity capture
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The peptide TMITPSLHACRSTLED, representing the
N-terminal 16 amino acids of the alpha-complementing factor
of beta-galactosidase encoded in pUCl9 (and also
representing the 16 constant N-terminal amino acids in all
of the peptides described in Example 1 above) is used to
raise a polyclonal rabbit antibody using standard
procedures.
A single ampicillin resistant E. coli colony
derived from the mini-library transformation described in
Example 1 is picked and induced lysates are prepared as
described in Example 1. A control lysate from cells with
nonrecombinant vector is prepared in parallel.
Immunoreactive proteins are precipitated from the lysates
by incubation of 1 ml aliquots with a 1:100 dilution of
antiserum followed by precipitation with Protein-A using
standard methods. The immunoprecipitate iswsuspended in 50
ul H20, and a 10 ul aliquot is suspended in 40 ul of
MALDI-matrix (~-cyano-4-hydroxycinnamic acid dissolved in
1:2 acetonitrile:l.5~ trifluoroacetic acid (ACCA), and 100
nL applied to the MS probe, air dried, and subjected to
matrix assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry using methods and procedures
well known in the art.
The mass spectrum of the immunoprecipitate from
the induced cell lysate of the clone under examination is
observed to contain a distinct peak, at a position
corresponding to a mass of 8485 3 Daltons, that is not
observed in the control. Comparison of the experimental
results with the values in the table in example 1 above
indicates that the insert begins at position 9241 of the
reference sequence and proceeds from left to right in the
Genbank sequence. Retrieval of the reference sequence
beginning at position 9241 indicates that the cloned
sequence begins with "GAATTCACATAAATCGCAAATTTTTTTTTCCTTCCC
AGAGCCATCCAAAACTCTGTTTGTCAAAGGCCTGTCTGAGGATACCACTGAAGAGAC
ATTAAAG..." {first 100 nucleotides shown). The
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identification is confirmed by dideoxy sequencing as
described in Example 1.
Example 3. Identification of a subcloned EcoRI fragment
of a clonedhuman ctene:analysis of peptides from multiple
reading frames.
The vector pTriplEx is digested with the
restriction endonuclease BglII and the ends of the
linearized plasmid are backfilled using Klenow fragment of
E. coli DNA polymerase I. The plasmid is treated with the
restriction endonuclease SmaI, blunt end ligated with DNA
ligase and transformed into competent E. coli BLR cells.
Ampicillin resistant colonies are selected on LB ampicillin
plates, and a single colony is picked and used to prepare
a plasmid miniprep. The plasmid, here named pTriplEx', is
linearized with EcoRI and a mini library is prepared using
as inserts the set of fragments produced by complete
digestion of the insert in EMBL3 human nucleolin clone
described in example 1. Competent E coli TOPP-1 cells are
transformed with the mini library and a single ampicillin
resistant colony is isolated. A 250 ml liquid culture of
cells from this colony is grown in LB-ampicillin medium at
degrees to a density of 2 x 108 cells per ml, induced
with 1 mM IPTG for 2 hours, concentrated to a volume of 10
ml by centrifugation, and lysed by sonication on ice with
25 six intermittent 30 second sonication pulses.
Control cells with nonrecombinant plasmid are prepared in
parallel. Immunoprecipitates of both lysates are prepared
as in Example 2.
An 10 ul aliquot of each immunoprecipitate is
suspended in 40 ul of MALDI-matrix and subjected to
MALDI-TOF mass spectrometry. The spectrum of the lysate
from the plasmid-containing cells is observed to contain
two distinct peaks not present in the control lysate, one
at a mass of 4254 2 Daltons and the other at a mass of
2635 2 Daltons.
To identify the nucleotide sequence adjacent to
the pTriplEx' vector, each EcoRI site in the J05584
sequence is identified and ligated, in silico, to the EcoRI
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site in the pTriplEx' vector. For each such in silico
construct, the amino acid sequences of the two expected
hybrid translation products (from each of the start codons
in the vector to the first in frame stop codons encountered
in the insert) are calculated. The mass of each peptide is
calculated and all 10 peptide pairs are tabulated, as shown
in the table below. Comparison of the experimental results
(i.e., peptides of 4255 and 2635 Da.) with the values
predicted in the table indicates that the insert begins at
position 4028 of the reference sequence and proceeds in the
forward direction. It is concluded that the 5' end of the
sequence joined to the vector is "GAATTCTCTTGGGTT
GTTGTGGTGTGCTAGACTTAATTACCCATGAATGATTTTGTCCTCTTGAGAAAATTT
CAATAGCACATCTATTAGTGTTTTTTAT..." (first 100 nucleotides
shown). The identification is confirmed by dideoxy
sequencing from the plasmid using a primer 150 nucleotides
3' to the pTriplEx' EcoRI site.
Position of Orientation
EcoRI site in pTriplEx', Start Codon Predicted Peptide Mass
3190 forward 1st 6137
3190 forward 2nd 5707
3190 reverse 1st 6278
3190 reverse 2nd 3891
4208 forward 1st 4255
4208 forward 2nd 2635
4208 reverse 1st 19748
4208 reverse 2nd 3905
6066 forward 1st 3595
6066 forward 2nd 3606
6066 reverse 1st 6401
6066 reverse 2nd 1363
9241 forward 1st 3583
9241 forward 2nd 7122
9241 reverse 1st 4582
9241 reverse 2nd 1746
9543 forward 1st 5306
9543 forward 2nd 1477
9543 reverse 1st 9906
9543 reverse 2nd 2516
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The mass values above are computed by translating
each hypothetical fusion polypeptide without the N-terminal
methionine that is removed in vivo in E. coli.
Example 4. Identification of a specific mutationin a
human gene.
Blood is drawn from a man and wife and from their
three children, and DNA is prepared from blood leukocytes
of each using standard methods. Two 20-nucleotide PCR
primers - one representing nucleotides 3190-3210 of the
nucleolin sequence described previously (the forward
primer) and the other representing the reverse complement
of nucleotides 4008-4028 (the reverse primer) - are used
to generate an 838 nucleotide PCR amplicon using high
fidelity thermostabile proofreading DNA polymerase. The
amplicon is cloned into the pTriplEx' vector described
previously, and 1000 transformant colonies from each
amplification are pooled to create five bacterial cultures,
two derived from the parents and three derived from their
offspring. Each bacterial culture is treated as described
in the previous example to produce five lysates and five
MALDI-TOF mass spectra. The spectrum from the father shows
two prominent peaks at positions corresponding to 6137 and
5707 Daltons. The same peaks are observed for the peptides
derived from two of the offspring. The mother and the
third child show not two peaks but three, two at 6137 and
5707 Da and a new one at 6169 Da. The new peak is 32 Da
bigger than the 6137 peak, consistent with a change from
valine to methionine with respect to the reference
sequence. The fact that there is no new peak derived from
the 5707 Da peak indicates that the base changes)
responsible for the valine-to-methionine substitution in
the larger peptide is silent with respect to the reading
frame encoding the 5707 Da. peptide. Of the six valine
codons in the 6137 Da. peptide, only one, the GTG codon at
position 3223, can be changed to give this result, the
change being a G to A transition (to ATG) at position 3223.
It is concluded that the mother and third child are
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heterozygous carriers for a single nucleotide polymorphism,
a G to A transition, at position 3223. Dideoxy sequencing
across the relevant region confirms this conclusion.
Example 5. Identification of a specific mutations in a
S human aene; analysis of pooled samples.
In this example known portions of the reference
sequence are used to design PCR primers, which are then
used to generate PCR products that are cloned, expressed in
fusion peptides, and analyzed in a parallel fashion. The
reference sequence predicts a peptide of a particular mass
and composition; deviations from the prediction indicate
differences in sequence from the reference sequence, in
this example single nucleotide polymorphisms.
Two oligonucleotide primers are synthesized using
standard methods. In one, CCCGAATTCAGCAGGTAAAAATCAAGG, the
first ten nucleotides contain an EcoRI site (underlined)
and last seventeen nucleotides correspond to the first
seventeen nucleotides of exon 2 of the human nucleolin
gene. The other, GGGGAATTCTTACTCTTCTCCACTGCTAT, the last
seventeen nucleotides correspond to the reverse complement
of the last seventeen nucleotides of exon 2, followed
immediately (in the sense orientation of the
oligonucleotide) by the stop codon TAA and a sequence that
includes an EcoRI site (underlined).
Blood is drawn from twenty individuals and PCR
amplicons are produced as described in the previous
example, using the two primers just described. The
amplicons are pooled and cloned into the EcoRI site of
pUCl9 as described in Example 2 above, and the bacterial
cultures are treated as described in Example 2 above to
produce a single MALDI-TOF mass spectrum derived from all
twenty pooled samples. The spectrum shows a major peak at
6873-3 Da., corresponding the predicted mass of the fusion
peptide encoded by the exon 2 reference sequence fused to
the vector peptide sequence, and two smaller peaks at
6862 3 Da. and 6915 3 Da. The amplitude of the 6862 peak
is approximately 1/20 of the 6872 peak, and the amplitude
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of the 6916 peak is approximately 1/40 that of the 6872
peak. The -10 Da. shift in the 6862 peak relative to the
6872 peak is that predicted for a single nucleotide
polymorphism (SNP) that produces a proline to serine
substitution in exon 2 that is already known to exist in
the human population at a frequency of approximately 5%,
and so it is concluded that in the forty haploid genomes
present in the twenty individuals, two copies of this
polymorphism are very likely present. The +44 Da shift in
the 6916 peak indicates an alanine to aspartic acid
substitution in exon 2 that was not previously known, and
that is present in one copy in the sample of forty haploid
genomes.
In this example the sample was heterogeneous
because amplicons from a number of individual individuals
were pooled prior to analysis. But the heterogeneity
could, in other cases, be intrinsic to a single sample.
For example, the sample could be a tumor biopsy containing,
for example, a mixture of cells that are heterogeneous with
respect to mutations in oncogenes or tumor suppressor
genes, and so PCR amplification of the oncogene or tumor
suppressor gene would yield a heterogeneous amplicon.
Example 7. Application of a comQuter program to
ctenerate a data set of mass shifts for all possible sincrle
nucleotide substitutions in a nucleotide sequence.
A computer program was written to compute the
mass shifts for all single nucleotide substitutions in a
nucleotide sequence. The program uses the amino acid mass
values given in the table below. The input to the program
is (1) a nucleotide sequence, and (2) a choice by the user
of which of the six possible reading frames (3 forward and
3 reverse) to be considered. The program translates the
input sequence and computes the masses of the encoded
peptides. It then generates all possible single nucleotide
substitutions of the sequence, computes a new set of
peptides, compares them to the original peptide(s), and
lists all of the mass differences between the mutant and
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non-mutant peptides. The program output is a listing of
the peptide mass changes for all possible single nucleotide
substitutions in the input sequence. The program then
accepts input representing the mass-shift threshold for
detection, i.e., the mass shift below which the shift is
treated as not detectable. Output is a listing of all
mutations in the sequence that are not detectable at the
set threshold.
Amino Acid S of Mass
Alanine A 71.08 Da
Arginine R 156.19
Asparagine N 114.10
Aspartic acid D 115.09
Cysteine C 103.14
Glutamic acid E 129.12
Glutamine Q 128.13
Glycine G 57.05
Histidine H 137.14
Isoleucine I 113.16
Leucine L 113.16
Lysine K 128.17
Methionine M 131.19
Phenylalanine F 147.18
Proline P 97.12
Serine S 87.08
Threonine T 101.10
Tryptophan W 186.21
Tyrosine Y 163.18
Valine V 99.13
Nonsense Z -
The program was run with the 24 nucleotide input
sequence CAACTAGAAGAGGTAAGAAACTAT. Two reading frames were
selected; the forward reading frame beginning with the
first nucleotide (F1) and the reverse (antisense) reading
frame beginning with the second antisense nucleotide (R2).
The results are shown below.
(begin]
Enter Sequence:
[input] CAACTAGAAGAGGTAAGAAACTAT
[output] Protein: QLEEVRNY
Which reading frames would you like to examine?
l: Forward (F1)
2: Forward; first base removed (F2)
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3: Forward; second base
removed (F2)
4: Reverse (R1)
5: Reverse first base removed
(R2)
6: Reverse second removed (R3)
[input] 1,5
[output]
MASS DIFFERENCES
Location Mutation Frame F1 Frame R2
None 1032.13 722.89
/A(K) 0.04 0.00
1 C- { G (E) 0 . 99 0 . 00
\T (Z) -1032 . 13 0 . 00
/G {R) 28 . 06 0 . 00
2 (Q) A- { T (L} -14 . 97 0 . 00
\C(P) -31.01 0.00
/G (Q) 0 . 00 0 . 00
3 A-{ T{H) 9.01 0.00
\C {H) 9 . Ol 0 . 00
/A (I ) 0 . 00 276 . 34
4 C-{ G(V) -14.03 276.34
\T (L) 0 . 00 0 . 00
/C(P) -16.04 299.37
5 (L) T- { A (Q) 14 . 97 226 . 32
\G(R) 43.03 200.24
/G(L) 0.00 241.29
6 A- { T {L) 0 . 00 241 . 33
\C(L) 0.00 242.28
/T(Z) -790.84 -34.02
7 G-{ C(Q) -0.99 -34.02
\A(K) -0.95 0.00
/G{G) -72.07 -60.10
8 (E) A-{ T(V) -29.99 16.00
\C(A) -58.04 -44.04
/G(E) 0.00 -34.02
9 A-{ T(D) -14.03 -34.02
\C(D) -14.03 -48.05
/T(Z) -661.72 0.00
10 G- { C (Q) -0 . 99 0 . 00
\A (K) -0 . 95 0 . 00
/G(G) -72.07 -16.04
11 (E) A-{ T(V) -29.99 23.98
\C (A) -58 . 04 43 . 03
/T(D) -14.03 0.00
12 G-{ C(D) -14.03 -14.03
\A(E) 0.00 34.02
/T(L) 14.03 -423.52
13 G-{ C(L) 14.03 -423.52
\A(I) 14.03 0.00
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/C(A) -28.05 -60.04
14 (V) T-{ A(E) 29.99 -16.00
\G(G) -42.08 -76.10
/G(V) 0.00 -26.04
15 A-{ T(V) 0.00 -49.08
\C (V) 0 . 00 -48 . 09
/G(G) -99.14 0.00
16 A- ~ T (Z) -433 . 47 0 . 00
\C(R) 0.00 0.00
/T(I) -43.03 76.0
17 (R) G-{ C(T) -55.09 16.06
\A(K) -28.02 60.10
/G(R) 0.00 10.04
18 A- { T (S) -69 . 11 14 . 02
\C(S) -69.11 -16.00
/G (D) 0 . 99 0 . 00
19 A- { T (Y) 49 . 08 0 . 00
\C(H) 23.04 0.00
/G(S) -27.02 -28.05
20 (N) A- f T(I) -0.94 15.96
\C (T) -13 . 00 -42 . 08
/A (K) 14 . 07 48 . 05
21 C-f G(K) 14.07 14.03
\T (N) 0 . 00 14 . 03
/C(H) -26.04 18.03
\G(D) -49.08 0.00
22 T-~ A(N) -48.09 0.00
/G(C) -60.04 -12.06
23 (Y) A-~ T(F) -16.00 15.01
\C (S) -76 . 10 43 . 03
/C(Y) 0.00 -14.03
24 T-{ A(Z) -163.18 0.00
\G(Z) -163.18 0.00
Enter
the detection
threshold;
[input] 0.8 Dalton.
[output] Undetectable amino acid substitutions:1.(Q)C-A(K)
The numbers in the first column denote each
nucleotide in the sequence. Note that for each nucleotide
in the input sequence there are three possible
substitutions, so that the number of lines in the output
data set is 72 (3 x 24). The amino acids encoded in each
F1 codon are shown in the second column, followed by all
possible single nucleotide substitutions at each position
in the fourth column. The fifth column shows the amino
acids encoded by the new codons, and the sixth column shows
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the mass change (if any) due to the amino acid substitution
(if any) or translation termination (if any) due to the
nucleotide substitution. The last column shows the mass
changes due to the same substitutions when translation is
in the R2 reading frame. The detection threshold value of
0.8 Daltons was entered; the program output indicated that
only one substitution, at position 1 in the encoded
peptide, would go undetected at this threshold value.
Note also that the expression of polypeptides
from two reading frames makes the analysis significantly
more robust than if just one reading frame is used. For
example, if just reading frame 1 is used, a shift of -14.03
Daltons could be due to an E-to-D substitution at amino
acid 3, or to an E-to-D substitution at amino acid 4, or to
an L-to-V substitution at amino acid 2. When the
additional reading frame data are considered, however, each
of these possibilities is distinguished from the others and
the ambiguity is thereby eliminated. Indeed, when up to
six reading frames are considered, there is little or no
ambiguity for the great majority of substitutions, even for
sequences as long as several hundred nucleotides.
A data set/database such as that generated above
can have great utility in the practice of the instant
invention when searched by a computer program that searches
the database using experimentally determined peptide mass
data. Many such programs can be generated. One example is
given below.
Enter reference sequence
Compute reverse complement of reference sequence
Translate beginning at each nucleotide
Translate beginning at each nucleotide
Create relational database of peptides
and nucleotide positions
Compute predicted masses of peptides; create relational
database of peptides, masses, and nucleotide positions
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Enter experimentally determined
mass data for peptides) derived
from unknown sequence
Search database for correspondence between entered
mass data and predicted mass values in database
Output location of unknown sequence
Example 8. Analysis of exon 2 of the human
rds /~eripherin gene .
The sequence of exon 2 of the human
rds/peripherin gene (Genbank accession M73531) is shown
below. Intron sequence is shown in lower case; exon
sequence in upper case.
gggaagcccatctccagctgtctgtttccctttaagTCGAATCAAGAGCAACGTGGA
TGGGCGGTACCTGGTGGACGGCGTCCCTTTCAGCTGCTGCAATCCTAGCTCGCCACG
GCCCTGCATCCAGTATCAGATCACCAACAACTCAGCACACTACAGTTACGACCACCA
GACGGAGGAGCTCAACCTGTGGGTGCGTGGCTGCAGGGCTGCCCTGCTGAGCTACTA
CAGCAGCCTCATGAACTCCATGGGTGTCGTCACGCTCCTCATTTGGCTCTTCGAGgt
aggccctgggcagctgggggtagagggtaaggagagcctcc
Two primers, of sequences
GGCCCGGAATTCTCCAGCTGTCTGTTTCCCTTTAAG and
AATTTACTCGAGCTACCCCCAGCTGCCCAGGGCCTAC were synthesized and
used to PCR amplify rds/peripherin exon 2 from an
individual known to carry a wild type allele of
rds/peripherin. The amplicon was cut with EcoRI and XhoI
and cloned into the EcoRI/XhoI sites of the pGEX derivative
described in Nelson et al. The resulting plasmid was cut
with Xho 1, treated with Klenow fragment of DNA polymerase,
and self-ligated to produce a construct expected to produce
a fusion protein with the sequence shown below.
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPY
YIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKD
FETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLD
AFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIQ
DLVPHTTPHHTTPHHTTPHHTTPQDLNSPAVCFPLSRIKSNVDGRYLVDGVPFSCCN
PSSPRPCIQYQITNNSAHYSYDHQTEELNLWVRGCRAALLSYYSSLMNSMGVVTLLI
WLFEVGPGQLGVARSSGRIVTD
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The same primers were used to amplify
rds/peripherin exon 2 from an individual known to carry a
mutation in the exon that removes a FinI restriction site.
An amplicon containing the mutation was cloned and
expressed as described above for the non-mutant sequence.
Cells containing both constructs were grown to
mid log phase in LB medium, induced with 1mM IPTG, and
incubated for 2 hours at 25~ Cells were collected by
centrifugation and extracted with B-per according to the
suppliers instructions. GST fusion proteins were purified
by standard methods for analysis by MALDI-TOF mass
spectrometry, which is performed as described previously.
The measured masses of the two fusion proteins
are 35571 Da and 35630. Da. The difference between the
two is 59 Da, indicative of a substitution of arginine for
proline in the peptide. Examination of the exon 2 sequence
reveals a Fin I site (GTCCC) whose last two nucleotides are
part of the first proline codon (CCT) in the sequence. It
is concluded that a proline-to-arginine substitution is
present at this proline. Tt is further concluded that the
codon very likely suffered a transversion at the second
position to create the arginine codon CGG. Dideoxy
sequencing across the exon 2 sequence in both constructs
confirms these conclusions.
Example 9. In vitro analysis of exon 2 of human
rds/peripherin.
The amplicons described in the previous example
are reamplified using the upstream primer
5'GGATCCTAATACGACTCACTATAGGGAGACCACCATGCATCACCATCATCACCAT
CACCACTCTCCAGCTGTCTGTTTCCCTTTAAG and the downstream primer
5' CTTAGTCATTATACCCCCAGCTGCCCAGGGCCTAC. The upstream
primer contains a T7 promoter followed by a translation
initiation sequence (start codon underlined) followed by a
sequence encoding eight histidines followed by sequence
identical to the red/peripherin sequence immediately 5' to
rds/peripherin exon 2. The downstream primer contains two
stop codons (in antisense orientation) preceding the
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sequence complimentary to the sequence just 3' to
red/peripherin exon 2.
The reamplification products are transcribed and
translated in a coupled cell free system (transcription by
T7 polymerase; translation by rabbit reticulocyte lysate)
using established methods and procedures. Immobilized
metal affinity chromatography is used to purify the
translation products, and the translation products are
analyzed by MALDI-TOF mass spectroscopy as in the previous
example. The two major translation products are observed
to differ by 59.1 Da, indicative of a substitution
of arginine for proline in the polypeptide. By logic
identical to that presented in the previous example, it is
concluded that that the polypeptides differ by a
proline-to-arginine substitution at the position of the
first proline of the exon-encoded sequence.
In the examples given above, the only physical
parameter whose value was measured was polypeptide mass.
It should be clear to the reader, however, that assessing
certain other polypeptide properties, such as amino acid
composition or amino acid sequence, may also serve to
locate an unknown sequence with respect to the reference
sequence. Such data might be obtained, for example, by
partial or complete digestion of the peptide, prior to
spectrometry, with endopeptidases such as trypsin,
chymotrypsin, or pepsin, or with aminopeptidases or
carboxypeptidases. Analysis can be performed with a
variety of spectrometric methods besides MALDI-TOF and ESI,
such as tandem mass spectrometry (MS/MS), quadripole time
of flight spectrometry (Q-TOF), or Fourier transform ion
cyclotron resonance (FTICR) mass spectrometry. Other
analytical methods well known in the art can also be used
to analyze the fusion peptides, such as gel or capillary
electrophoresis or high performance liquid chromatography
(HPLC). It should also be clear that the instant invention
has utility even if it does not unambiguously assign an
unknown sequence to just one place in the reference
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sequence. For example, a search might eliminate all but
four positions in the reference sequence, each on a
different chromosome; if the chromosomal location of the
unknown sequence were known from some independent
determination, such as fluorescence in situ hybridization
(FISH), then the assignment could be made unambiguous.
Likewise, there may be circumstances where the reference
sequence is complex, representing, for example, an
annotated combination of sequences derived from more than
one individual, strain or species, which could be viral,
procaryotic or eucaryotic: In such circumstances, the
instant invention could be used, in medical, forensic or
population biology contexts for example, to determine the
individual, strain, or species from which the unknown DNA
originated, or, conversely, it could be used to rule out an
individual , strain or species as the source of origin of
the unknown DNA.
Some embodiments of the invention include
multiplex or pooled-sample analysis wherein peptides
encoded in more than one DNA fragment are co-analyzed. For
example, peptides encoded in more than one exon of a gene
may be combined and analyzed in concert, or samples from
multiple individuals may be pooled and analyzed together.
Some embodiments of the invention include methods
for determining the sequence of a polynucleotide,
comprising providing a nucleic acid fragment having
homology to a known reference sequence; expressing at least
one polypeptide from said fragment; and assessing at least
one physical property of said at least one polypeptide to
determine the sequence of said fragment by comparing said
at least one property to the predicted properties of
polypeptides encoded in said known reference sequence. The
method also includes wherein said nucleic acid fragment
contains a difference with respect to the reference
sequence wherein said difference is selected from the group
consisting of single nucleotide polymorphism, single
nucleotide substitution, single nucleotide deletion, single
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nucleotide insertion, multiple nucleotide substitution,
multiple nucleotide deletion, multiple nucleotide
insertion, DNA duplication, DNA inversion, DNA
translocation, and DNA deletion/substitution. The method
further includes embodiments wherein said nucleic acid
fragment comprises an exon or a cDNA. The method further
includes embodiments wherein the polypeptide(s) contain
heterologous epitope tags and expressed in living cells or
expressed in a cell free systems such as an E. coli
extract, rabbit reticulocyte extract, or wheat germ
extract. The invention further includes embodiments
wherein the peptides. are purified by a variety of methods
including gel electrophoresis, capillary electrophoresis,
liquid chromatography (LC), capillary liquid
chromatography, high performance liquid chromatography
(HPLC), differential centrifugation, filtration, gel
filtration, membrane chromatography, affinity purification,
biomolecular interaction analysis (BIA), ligand affinity
purification, glutathione-S-transferase affinity
chromatography, cellulose binding protein affinity
chromatography, maltose binding protein affinity
chromatography, avidin/streptavidin affinity
chromatography, S-tag affinity chromatography, thioredoxin
affinity chromatography, metal-chelate affinity
chromatography, immobilized metal affinity chromatography,
epitope-tag affinity chromatography, immunoaffinity
chromatography, immunoaffinity capture, capture using
bioreactive mass spectrometer probes, mass spectrometric
immunoassay, and immunoprecipitation. The method further
includes embodiments wherein the physical property that is
determined is mass, and wherein mass is determined by a
variety of methods including mass spectrometry, MALDI-TOF
mass spectrometry, electrospray ionization mass
spectrometry (ESI) ) tandem mass spectrometry (MS/MS),
quadripole time of flight spectrometry (Q-TOF), Fourier
transform ion cyclotron resonance (FTICR) mass
spectrometry, gel electrophoresis, capillary
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electrophoresis, and high performance liquid chromatography
(HPLC). The method further includes embodiments wherein
the physical property that is assessed is partial or
complete amino acid composition or sequence.
In another embodiment the present invention
includes a method for genetic analysis comprising providing
a nucleic acid fragment, expressing at least one
polypeptide from the fragment, and assessing at least one
physical property of said at least one polypeptide to
determine the coding capacity of said fragment by comparing
said at least one property to the predicted properties of
polypeptides encoded.in a known reference sequence. In a
further embodiment the invention includes method for
analyzing fragments that contain a differences with respect
to the reference sequence that include of single nucleotide
polymorphisms, single nucleotide substitutions, single
nucleotide deletions, single nucleotide insertions,
multiple nucleotide substitutions, multiple nucleotide
deletions, multiple nucleotide insertions, DNA
duplications, DNA inversions, DNA translocations, and DNA
deletion/substitutions. Tn further embodiments the
invention includes methods for analyzing nucleic acid
fragment representing exons or cDNAs, for examining
polypeptides that carry epitope tags, for examining
polypeptides expressed in living cells or in cell free
systems such E. coli extracts, rabbit reticulocyte
extracts, and wheat germ extracts. The invention further
includes embodiments wherein the peptides are purified by
a variety of methods including gel electrophoresis,
capillary electrophoresis, liquid chromatography (LC),
capillary liquid chromatography, high performance liquid
chromatography (HPLC), differential centrifugation,
filtration, gel filtration, membrane chromatography,
affinity purification, biomolecular interaction analysis
(BIA), ligand affinity purification,
glutathione-S-transferase affinity chromatography,
cellulose binding protein affinity chromatography, maltose
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binding protein affinity chromatography,
avidin/streptavidin affinity chromatography, S-tag affinity
chromatography, thioredoxin affinity chromatography,
metal-chelate affinity chromatography, immobilized metal
affinity chromatography, epitope-tag affinity
chromatography, immunoaffinity chromatography,
immunoaffinity capture, capture using bioreactive mass
spectrometer probes, mass spectrometric immunoassay, and
immunoprecipitation. The method further includes
embodiments wherein the physical property that is
determined is mass, and wherein mass is determined by a
variety of methods including mass spectrometry, MALDI-TOF
mass spectrometry, electrospray ionization mass
spectrometry (ESI) ) tandem mass spectrometry (MS/MS},
quadripole time of flight spectrometry (Q-TOF}, Fourier
transform ion cyclotron resonance (FTICR) mass
spectrometry, gel electrophoresis, capillary
electrophoresis, and high performance liquid chromatography
(HPLC). The method further includes embodiments wherein
the physical property that is assessed is partial or
complete amino acid composition or sequence.
In additional embodiments, the invention includes
methods for assessing a disease, condition, genotype, or
phenotype comprising providing a nucleic acid fragment from
a biological sample, and expressing at least one
polypeptide from said fragment, and assessing at least one
physical property of said at least one polypeptide to
determine the sequence of said fragment by comparing said
at least one property to the predicted properties of
polypeptides encoded in a known reference sequence, and
correlating said determined sequence with said disease,
condition, genotype or phenotype. The biological sample
may be obtained from a virus, organelle, cell, tissue, body
part, exudate, excretion, elimination, or secretion of a
healthy, diseased or deceased microorganism, protist, alga,
fungus, animal or plant.
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Other embodiments include diagnostic or
prognostic tests for diseases, conditions, genotypes, or
phenotypes comprising providing a nucleic acid fragment
from a biological sample, and expressing at least one
polypeptide from the fragment, and assessing at least one
physical property of one or more of the polypeptides to
determine the sequence of the fragment by comparing the
property or properties to the predicted properties of
polypeptides encoded in a known reference sequence. The
sample may be obtained from a virus, organelle, cell,
tissue, body part, exudate, excretion, elimination, or
secretion of a healthy, diseased or deceased microorganism,
protist, alga, fungus, animal or plant. In further
embodiments, the test may detect heterozygote status, and
it may indicate responses to drug or therapeutic
treatments. The test may be for a genetic disease such as
Alzheimer's disease, Ataxia talangietasia (ATM), Familial
adematous polyposis (APC), Hereditary breast/ovarian cancer
(BRCAl, BRCA2), Hereditary melanoma (CDK2, CDKN2),
Hereditary non-polypsosis colon cancer (hMSH2, hMLHl,
hPMSl, hPMS2), Hereditary retinoblastoma (RB1), Hereditary
Wilm's Tumor (WT1), Li-Fraumeni syndrome (p53), Multiple
endocrine neoplasia (MEN1, MEN2), Von Hippel-Lindau
syndrome (VHL), Congenital adrenal hyperplasia, Androgen
receptor deficiency, Tetrahydrobiopterin deficiency,
X-Linked agammaglobulinemia, Cystic Fibrosis (CFTR),
Diabetes, Muscular Dystrophy (DMD, BMD), Factor X
deficiency, Mitochondrial gene deficiency, Factor VII
deficiency, Glucose-6-Phosphate deficiency, Pompe Disease,
Hemophilia A, Hexosaminidase A deficiency, Human Type I and
Type III Collagen deficiency X-linked SCID, Retinitis
pigmentosa (RP) LIACAM deficiency, MCAD deficiency, LDL
Receptor deficiency, Ornithine Transcarbamylase deficiency,
PAX6 Mutation Phenylketonuria, RB1 Gene Mutation, Tuberous
Sclerosis, von Willebrand Factor Disease, Werner syndrome,
cancer, or an infectious disease.
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Further embodiments include methods for assessing
a disease, condition, genotype, or phenotype providing a
nucleic acid fragment from a biological sample, and
expressing at least one polypeptide from the fragment,
assessing at least one physical property of one or more of
the polypeptides to determine the coding capacity of the
nucleic acid fragment by comparing said at least one
property of the polypeptide(s) to the predicted properties
of polypeptides encoded in a known reference sequence, and
correlating said determined sequence with said disease,
condition, genotype or phenotype. The biological sample
may obtained from a.virus, organelle, cell, tissue, body
part, exudate, excretion, elimination, or secretion of a
healthy, diseased or deceased microorganism, protist, alga,
fungus, animal or plant. The particular original source
w- may be blond, sweat, tears, urine, semen, saliva, sweat,
feces, skin or hair, or it may come from the environment
that the living inhabits or has inhabited, such as air,
soil or water.
Further embodiments include diagnostic or
prognostic tests for a disease, condition, genotype, or
phenotype selecting a nucleic acid fragment taken from a
virus, organelle, cell, tissue, body part, exudate,
excretion, elimination, or secretion of a healthy, diseased
or deceased microorganism, protist, alga, fungus, animal or
plant, expressing at least one polypeptide from the
fragment, assessing at least one physical property of the
polypeptide(s) to determine the coding capacity of the
fragment by comparing the property or properties to the
predicted properties of polypeptides encoded in a known
reference sequence. The particular original source of the
nucleic acid may be blood, sweat, tears, urine, semen,
saliva, sweat, feces, skin or hair, or it may come from the
environment that the living inhabits or has inhabited, such
as air, soil or water. The test may detect heterozygote
status or indicate or response to a therapeutic drug or
treatment. It may detect genetic disease, such Alzheimer's
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disease, Ataxia talangietasia (ATM), Familial adematous
polyposis (APC), Hereditary breast/ovarian cancer (BRCAl,
BRCA2), Hereditary melanoma (CDK2, CDKN2), Hereditary
non-polypsosis colon cancer (hMSH2, hMLHl, hPMSl, hPMS2),
Hereditary retinoblastoma (RB1), Hereditary Wilm's Tumor
(WT1), Li-Fraumeni syndrome (p53), Multiple endocrine
neoplasia (MEN1, MEN2), Von Hippel-Lindau syndrome (VHL),
Congenital adrenal hyperplasia, Androgen receptor
deficiency, Tetrahydrobiopterin deficiency, X-Linked
agammaglobulinemia, Cystic Fibrosis (CFTR), Diabetes,
Muscular Dystrophy (DMD, BMD), Factor X deficiency,
Mitochondrial gene deficiency, Factor VII deficiency,
Glucose-6-Phosphate deficiency, Pompe Disease, Hemophilia
A, Hexosaminidase A deficiency, Human Type I and Type III
Collagen deficiency X-linked SCID, Retinitis pigmentosa
(RP) LIACAM deficiency, MCAD deficiency, LDL Receptor
deficiency, Ornithine Transcarbamylase deficiency, PAX6
Mutation Phenylketonuria, RB1 Gene Mutation, Tuberous
Sclerosis, von Willebrand Factor Disease, and Werner
Syndrome, cancer, or infectious disease.
The invention further includes various
polypeptides that are created in the embodiments described
above.
Further embodiments of the invention take the
form of data a useful for detecting and analyzing DNA
mutations and polymorphisms stored in a physical medium in
computer readable form a plurality of DNA sequence
fragments contained within a reference DNA sequence, and
the sequences of the polypeptides encoded in said DNA
sequence fragments, the predicted sequences of a plurality
of polypeptides encoded in a set of transformed DNA
sequence fragments, each member of said set comprised of a
DNA sequence related to said DNA sequence fragment by a
specific change selected from the group consisting of
single nucleotide polymorphism, single nucleotide
substitution, single nucleotide deletion, single nucleotide
insertion, multiple nucleotide substitution, multiple
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nucleotide deletion, multiple nucleotide insertion, DNA
duplication, DNA inversion, DNA translocation, and DNA
deletion/substitution; b. means for comparing the predicted
sequences of said plurality of polypeptides with a test
sequence to determine identity of the test sequence with a
predicted sequence.
Additional embodiments include computer data
structures, comprising: data storage media; and data sets
in computer readable form on the data storage media
representing a plurality of polypeptide fragments of
polypeptides encoded by a reference polynucleotide
sequence; and second data sets in computer readable form on
the data storage media representing physical properties of
each of the polypeptide fragments; and means for
correlating empirically derived physical properties of test
polypeptides with second data sets to determine the
identity of the test polypeptides. The data structures may
further comprising third data sets in computer readable
form on said data storage media representing polynucleotide
fragments encoding the polypeptide fragments; and means for
correlating the identity of the test polypeptides with
polynucleotide fragments represented in the third data
sets. In these data the physical properties may include
mass or partial or complete amino acid composition or
sequence.
In yet additional embodiments, the invention
includes data structures in which reference polynucleotides
have a reading frame, and wherein one data set represents
polypeptide fragments encoded in frame and polypeptide
fragments encoded out of frame with respect to said
reference polynucleotide.
Further embodiments include computer implemented
methods for ascertaining the identity of nucleic acid
fragments encoding polypeptides, wherein the nucleic acid
fragments are fragments of known reference sequences,
comprising the steps of measuring a physical property of a
polypeptide comparing, in a computer, the measured physical
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property with a data set representing the predicted
corresponding physical properties of possible polypeptides
that are encoded by fragments of the reference sequence
within a predetermined size range; and identifying a match
between the measured physical property and a predicted
physical property in the data set; and displaying or
recording the results of the identifying step. The data
set may includes physical properties of polypeptides
encoded by in-frame and any of six out-of-frame fragments
of said reference polynucleotide.
Additional embodiments of the invention include
relational data sets .useful for detecting and analyzing DNA
mutations and polymorphisms comprising a plurality of DNA
sequence fragments contained within a reference DNA
sequence, the sequences of the polypeptides encoded in said
DNA sequence fragments, and the predicted sequences of a
plurality of polypeptides encoded in a set of transformed
DNA sequence fragments, each member of said set comprised
of a DNA sequence related to said DNA sequence fragment by
a specific change selected from the group consisting of
single nucleotide polymorphism, single nucleotide
substitution, single nucleotide deletion, single nucleotide
insertion, multiple nucleotide substitution, multiple
nucleotide deletion, multiple nucleotide insertion, DNA
duplication, DNA inversion, DNA translocation, and DNA
deletion/substitution. Further embodiments include
computer programs that search of these data sets.
The computer-implemented methods of the present
invention can be carried out on a general purpose computer,
such as, for example, a PC running the Windows, NT, Unix,
or Linux operating systems, or a Macintosh personal
computer. For some embodiments of the invention, a more
powerful computer mainframe would be desirable. Suitable
computers typically have a central processor, computer
memory (such as RAM), and a storage medium, such as a
floppy disk, a fixed disk or hard drive, a tape drive, an
optical storage medium such as a CD, DVD, or WORM drive, a
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removable disk, or the like, which can store data in
computer-readable form. Such computers typically have a
means, such as a monitor, for displaying data or
information, and are capable of storing program-generated
data in RAM or in the storage medium. Such computers can
also advantageously be connected to a printer, for
providing a fixed record of information generated by the
program.
A general purpose computer utilized in the
present invention could be programmed with a specific
program of the type described herein. In particular, this
program would generate data sets of all possible nucleotide
fragments, in all possible frames and in both orientations.
It would predict and store data sets reflecting the
translation products of those fragments. It would also
store, in a correlatable manner, a data set reflecting a
physical property (such as molecular weight) of each of
those fragments. One program that could be used in the
present invention would compare an empirically determined
physical property of a polypeptide translated from a
polynucleotide fragment from a biological sample with the
data set to determine, for example, which possible
polypeptide fragment or which possible polynucleotide
fragment corresponds to the sample. In this manner, the
identity of DNA in the sample can be determined.
In one embodiment, information directly or
indirectly related to the identity of the polynucleotide
fragment from the sample can be displayed, printed, and/or
stored. This can include the exact identity or sequence of
the polynucleotide, or a tag, label, or name associated
therewith. It could also be a diagnosis of a disease,
condition, genotype, or phenotype associated with that
particular polynucleotide.
CONCLUSION, RAMIFICATIONS AND SCOPE OF INVENTION
In conclusion, the invention specified here
provides a novel method for analyzing cloned DNA segments
and for identifying and/or assaying known or new
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polymorphisms or mutations in those DNA segments. The
method has unique and highly useful advantages over all
other methods the prior art.
The specific description of my invention
presented above should not be construed as limiting its
scope but rather as exemplification of certain embodiments
thereof. Many other variations and applications are
possible and can be practiced by one skilled in the art.
For the purpose of expression, multiple promoters and
translation start sites can be placed in the known
sequence, on one or both sides thereof, so that the unknown
sequence is translated in up to six different reading
frames. Or the unknown sequence can be a PCR amplicon that
is cloned into a vector in both orientations, thereby
yielding a mixture of clones, some translated from one
strand --and some from the other. Or promoters and-
translation start signals can be incorporated near one or
both ends of a transposable element, such as Tn3, Tn5, Tn7,
TnlO, Ty, P-element, and Mariner; of a virus such as herpes
virus, adenovirus, adeno-associated virus; or of a
retrovirus. Fusion protein expression need not take place
in bacteria, as in the examples given here, but may take
place in eucaryotic cells such as yeast or mammalian cells,
and cell free expression need not take place in a rabbit
reticulocyte lysate, as in the example, but in other cell
free systems. Other modalities for peptide capture can be
used, such as incorporating biotinylated lysine in the
peptides and capturing with avidin or streptavidin.
Additionally, protease recognition sites may be
incorporated into the known sequence to aid in fragment
preparation, such as placing an enterokinase cleavage site
and a poly-histidine sequence upstream of the junction to
the unknown sequence so that a peptide for analysis can be
released by enterokinase treatment of an affinity captured
polypeptide. Further, the DNA polymorphisms that are
identified and/or detected need not be limited to single
nucleotide polymorphisms, as in the examples, but could be
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of many other kinds such as microsattelite repeats of
different lengths or specific single nucleotide deletions,
single nucleotide insertions, multiple nucleotide
substitutions, multiple nucleotide deletions, multiple
nucleotide insertions, DNA duplications, DNA inversions,
DNA translocations, DNA deletion/substitutions or other
chromosomal rearrangements.
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