Note: Descriptions are shown in the official language in which they were submitted.
S;304
1 A TEST FOR TlIE DETE~`IINATIO~l OF PATERNITY ?~D FOR
TI~E ESTABLISHr~ENT OF I~DIVI~UAL GENETIC IDENTITY
This invention relates to a new and improved
10 diagnostic test applied to the determination of paternity and
for the establishment of individual genetic identityO It
should be noted that~although, as is the practice in the art,
a test is referred to as a paternity test; there is nothing
which precludes its employment in cases of disputed
15 maternity.
There are numerous situations when the ability to
determine an individual's identity is of importance; for
example, the matching of physical evidence left at the scene
20 of a crime with a particular suspect, the establishing of the
identity of an individual in relation to his/her mother or
father as in the determination of paternity or more generally
when establishing the genetic identity of a strain of a
virus, bacterium, alga, fungus, plant or animal. Some of the
tests employed for such determinations rely upon the
identification of polymorphic proteins in the plasma, from
the surface, or extracted from within the cells of the
individuals in question.
The well known human ABO blood group substances may
3o be used by way of explanation. The ABO blood group
substances are carbohydrate in composition and are
svnthesized by enzymes which are the products of a single
human gene. One form of the gene (the A allele) produces an
enzyme used in the synthesis of A-type blood, while another
form of the gene (the B allele) produces an enzyme used in
the synthesis of B-type blood. The absence of both alleles
~2~530~
1 lresults in the production of O-type blood, while the
presence of both alleles results in the production of AB-type
blood. The ABO substances possess antigenic properties and
may be detected immunologically by reaction with the
5 appropriate antisera. It is the differential reac~ivity of
these substances with said antisera which forms the basis of
the A, B, O and AB blood type groupings.
If everyone possessed the same blood type, the
substance would be useless in discriminating among
lO individuals. The fact that the blood group substances exist
in several forms (i.e., are polymorphic) allows for
discrimination. However, in terms of its power to exclude,
as in cases of disputed paternity, not only is the number of
different alleles important, but also the frequencies with
15 which those alleles occur. Since these allele frequencies
vary among populations, the efficacy of exclusion also
varies. The power of a test to exclude is represented by its
exclusion capability, a numerical value ranging from 0 to
1Ø The exclusion capability of the ABO system among
20 American blacks is .1774 while among American Caucasians it
is .1342. The exclusion capability increases to .1830 for
Swedes and to .1917 for Japanese.
One approach to increase the exclusion capability
has been to expand the analysis to include other polymorphic
substances. In Sweden, twelve polymorphic substances are
analyzed. The overall exclusion capability of this battery
of tests approaches .870. The addition of more systems to
the set, even if highly informative, will not increase the
cumulative probability greatly, once the number of systems
3o already involved is large. A survey of 25 systems based on
immunological tests (Antigen-Antibody reactions) revealed a
cumulative probability of non-paternity of .7694 while a
12~53Q~
l similar analysis of 32 systems based on biochemical tests
(enzyme reactions or electrophoretic mobility) yielded a
value of .9512. The combined 57 systems still only yielded
an exclusion value of .9887. Extensive investiyations are
5 not practical in terms of a paternity testing program since
many of the systems, because of cost, paucity of reagents,
technical complexity, low reliability, and/or insufficient
experience are not considered suitable for routine work.
It is well known in the forensic sciences to employ
lO multiple test systems for the determination of identity. For
example, in addition to the AB0 blood group antigens, MN and
Rh antigens are also analyzed. If the test sample is liquid
Le and Se antigens may also be included. Three red blood
cell enzymes acid phosphatase, phosphoglucomutase, and
15 esterase D are examined for the presence of electrophoretic
variants. Finally, tests for serum proteins such as
haptoglobins are also employed. As was the case for the
determination of paternity, the extent of these forensic
investigations is also limited by cost, technical complexity
and low reliability.
The above practical considerations not
withstanding, a more serious theoretical problem plagues all
of the existing tests. Since the tests are based on the
analysis of a protein or its activity, it is the gene product
and not the gene itself which is the subject of the
investigation. In accord with the instant inventions
disclosed hereinafter, it is preferable to analyze the gene
directly rather than the product of its expression, in
situations where paternity is of interest, because of the
3o degeneracy that is inherent in the process by which genetic
information is expressed.
~S3~
l The flow of genetic information in cells is well
known. The information directing the biosynthesis of any
protein is encoded in the sequences of DNA nucleotides known
as a gene. The DNA of the cell may be viewed as the storage
5 form of the genetic information. The DNA molecules are
large, chemically stable, easily replicated and contain many
gene sequences. For example, the entire genetic repertoire
of the bacteria E. coli is contained in a single DNA molecule
composed of approximately 4.2x106 nucleotide base pairs.
Transcription is the process by which the retrieval
of information is begun. Transcription involves the
resynthesls of the informatlon in the form of a nucleic acia
called ~NA. O~e type of RN~, messenger ~NA ~mR~A~,
transports the information to the site of protein synthesis
called ribosome.
Once the mRNA is synthesized from the gene the
process of protein synthesis ma~ begin. This process is
essentially one of molecular decoding, in which the
nucleotide sequence of the mRNA provides a template for the
synthesis of a particular protein. Since there is a change
from a nucleic acid language into that of a protein language,
this process of protein synthesis appropriately is referred
to as translation. Continuing the analogy a bit further, it
would be appropriate to think of the constituents of the
nucleic acids, the nucleotides, as representing the alphabet
of the nucleic acid language and the amino acids, the
building blocks of proteins, as representing the alphabet of
the protein language. During the process of translation not
only are the languages changing but the alphabets are
3o changing as well. This is a particularly complex process
which is known to involve over 100 types of molecules. As
the mRNA is passed through the ribosome (much like the tape
--5--
~21S3~4
1 through a tape recorder) groups of 3 nucleotides (codons) are
positioned such as to orient accessory RNA molecules, known
as transfer RNA (tRNA), carrying a single amino acid into the
proper alignment for the addition of the amino acid to the
5 growing protein chain.
Of special interest with respect to the subject
invention is the coding ratio of nucleotides to amino acids.
As mentioned above this ratio is three nueleotides coding for
one amino acid. Since it is neeessary to eode for twenty
10 different amino acids uniquely with the available four types
of nucleotides ~A, U, G, C~, three represents the minimum
accepta~le rati~. A coding ratio of one nuc~eotide to one
amino acid would only accomm~date f~ur of the twenty amino
aeids necessary for protein synthesis. A coding ratio of two
15 yielding 16 (42) eombinations likewise falls short of the
required eomplexity. However, with a eoding ratio of three,
64 (4 ) different eombinations are possible. This excess
of twenty eode words eonfers upon the genetic code a
eondition known as degeneraey. A degenerate eode contains
several different code words for the same amino aeid. The
situation does not exist, however, where one eode word would
speeify two different amino acids. The code may be
degenerate, but it is not ambiguous.
Knowing the sequence of nucleotides of a messenger
RNA, it is possible to explicitly write the sequence of amino
aeids eoded therein, but the reverse is not true. Because of
the degeneracy of the genetie eode, a number of nucleotide
sequences would be eonsistent with a given amino acid
sequence. For example, consider the fragments of a mRNA from
3o the same gene in two different individuals "A" and "B".
INDIVIDUAL "A" INDIVIDUAL "B"
mRNA [UUC CCC CGA GUU CUA AAG] [UUU CCG AGG GUC CUU AAG]
protein [phe-pro-arg-val-leu,lys] lphe-pro-arg-val-leu-lys]
lZlS3Q4
1 An analysis of the protein would indicate the two
individuals are identical, whereas an analysis of the mRNA
sequence would indicate clear differences. Any paternity
test based on a protein analysis be it either immunological
5 or biochemical would fail to distinguish between the two
individuals. A test based on the analysis of the genetic
material, either ~A, or pre~erably ~N~, w~u~d allow such a
distinction to be made.
Although the discussion above has centered on
10 determination of paternity in humans it should be kept in
mind that such tests, given the appropriate reagents, may be
extended to include certain other animal species (e.g.,
horses, cows, dogs, etc.). In reference to the subject
invention, because of the unique approach taken therein, the
15 test procedure is applicable to a determination of parentage
in any group of sexually reproducing organisms including
plants as well as animals.
In a further application of the subject invention,
the genetic identity of individuals may be established. This
20 application is particularly useful in the area of forensic
science or for the identification of strains of
microorganisms, plants or animals.
The object of this invention is to provide a new
and improved test for the determination of paternity in
sexually reproducing organisms and to establish individual
cenetic identity. These objectives are achieved by analyzing
the DNA of said organism in respect to one or more
~olymorphic genetic regions, differentiating the
30 ?olymorphisms in terms of relative size of the genetic
regions and by so doing characterize an individual member of
the species.
~S3~ ,
l In one embodiment, DNAs derived from the offspring,
the mother and for example the putative father are separately
digested with one, or more, restriction enzymes and the
resulting fragments are separated on the basis of size by
5 causing them to migrate through a gel matrix under the
influence of an electric current. The polymorphisms are
detected by hybridizing the ab~e-treated D~As with labe~led
(e.g., radioactive) "probe" DNAs.
The probe DNAs are variable D~A fragments that ha~e
lO been joined to a vector DNA which is able to replicate in a
host cell (e.g., plasmid pBR322, bacteriophage lambda or ~13
in Escherichia coli, or SV40 in monkey cells) and then
purified from the host cells.
The reacted probe DNAs allow visualization of the
15 position, and thus the sizes, o~ the DNA fragments of the
offspring, the mother, and the putative father, whose
sequences are homologous to those of the probe DNAs. Because
the probe DNAs have been chosen on the basis of their being
one allele from a polymorphic locus, the sizes of the DNA
fragments homologous to those of the probes will vary among
individuals.
All DNA fragments possessed by the offspring will
be derived from either the offspringls mother or father,
barring mutations or certain other rare events. A comparison
Of the sizes of the DNA fragments detected by the probe DNAs
thus allows one to determine whether or not the putative
father could be the biological father. For example, if the
offspring's DNA yields a ~600 hase-pair ~ragment homolog~us
to one of the pro~e DNAs, and if the mother's DNA lacXs this
3o fragment, then the biological father's DNA must contain it.
If the putative father's DNA lacks this fragment he can be
excluded as the biological father.
~S;~4
1 In a further embodiment, samples of DNAs derived
from a suspect and from physical e~Tidence (blood, s~in,
sperm, etc.) at a crime scene may be compared by the use of
the probes described above to establish identity bet~een the
5 samples and the suspect. Thus the DNA polymorphism with
respect to the hybridization assay provides the forensic
scientist with a "molecular fingerprint" to be included along
with the rest of the analysis of physical evidence.
In yet another embodiment, a sample of DNA deri~ed
from an individual is compared with that DNA derived from
other members of a strain of organism on the basis of
relative size for the purpose of establishing the strain
identity or said individual.
Description of the Drawinys
Figure 1 represents the autoradiograph described in
~xample VII.
Figure 2 represents the autoraaiograph as described
in Example VIII.
Figure 3 represents the autoradiograph as described
in Example IX.
In one of its embodiments, the instant invention
consists of the four interrelated steps of: DNA isolation
and restriction; gel electrophoresis and DNA blotting;
hybridization and washing; and finally autoradiography.
DNA Isolation and Restriction
-
The isolation of DNA from cell samples is carried
out ~y art recognized procedures. DNA preparation involves
cell lysis, sodium dodecyl sulfate and sodium perchlorate,
3o chloroform/isoamylalcohol extractions, and ethanol
precipitation.
_9_
12~S3()4
l Following its preparation each DNA sample is
subjected to analysis with one or more restriction
endonucleases. Restriction endonucleases are enzymes which
recognize short specific sequences of ahout 4-7 nucleotide
5 base pairs and cleave the DNA at or near these sites.
Although there are more than 200 restriction enzymes from
which to choose, the selection of any particular enzyme to
employ in the test would depend on the type of sample DNA ,
the number of fragments required and the availability and
lO cost of the reagents.
The human genome, which consists of approximately
6x109 base pairs of DNA, would be cleaved into 106 to
107 discrete fragments ranging in size from 102 to 105
base pairs by a single restriction endonuclease. The
15 complexity of such a digest is a reflection of the number and
location of the endonuclease specific cleavage points within
the sample DNA. An exhaustive identification of each
fragment from parallel treatments involving a number of
different endonucleases would, in theory, result in a
"molecular fingerprint" which would be unique for each human
being, Although theoretically possible, such a detailed
analysis is impractical. The subject invention overcomes
this problem by permitting the analysis of a subset of the
existing cleavage products. Employing the jargon of the
genetic engineer's art, one is said to "probe" the existing
cleavage products for the existence of the unique nucleotide
sequence of interest. One well known method for
accomplishing this analysis is the technique of ~outhern
blotting.
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--10--
~.2153~4
l Gel ~lectrophoresis and Blotting
According to the method of Southern (J. Mol. Biol.
~8:503-17 (197S)) the double stranded DNA ~ragments obtained
from the treatment with the restriction endonuclease are
5 separated by size by electrophoresis in an agarose gel, and
the DNA made single stranded by soaking the gel in alkali.
The gel is placed flat onto a "wick" of filter paper that
connects with a trough containing concentrated salt solution.
A single sheet of cellulose nitrate is then placed
lO on top of the gels and a large stack of dry absorbent paper
towels laid flat on top of the cellulose nitrate. The salt
solution is drawn up by the absorbent paper towels, passing
through the gel and cellulose nitrate sheet. As the solution
passes through the gel, the single stranded DNA will be
15 leached from the gel and pass onto the cellulose nitrate
filter. Cellulose nitrate has the property of binding
single-stranded DNA, so all the DNA will be leached from the
gel and pass onto the cellulose nitrate filter, Cellulose
nitrate has the property of binding single-stranded DNA, so
all the DNA will adhere to this support. The end result of
this procedure will be a perfect replica of the DNA from the
original agarose gel, but the DNA is now single-stranded and
immobilized on the cellulose nitrate filter sheet. The DNA
size pattern from the original agarose gel is, nevertheless,
faithfully preserved. Fragment sizes may be calibrated by
comparison to marker DNA of known sizes.
Hybridization and Washing
A hybridization reaction is said to occur when two
single-stranded DNAs from different sources reassociate to
3o
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12153~4
1 form a double-stranded DNA owing to complementary base
pairing b~tween the two interacting strands. DNA/RNA hybrids
may also be formed by means of the analogous associations.
With respect to the subject invention a DNA/DNA
5 hybridization is performed. One contributory source of
material for the hybridization reaction is the single-
stranded DNA present in the Southern ~lots of the restriction
fragments. The other sources of hybridizing strands are the
so-called "probe" DNAs. These DNAs represent variable DNA
10 fragments chosen on the basis that they represent sequences
corresponding to one allele of a polymorphic gene locus. A
full description of the isolation and characterization of the
"probes" is presented in a subsequent section of this
disclosure.
A variety of hybridization conditions are recog-
nized in the art including 50 percent formamide at 40-50~C or
moderate salt at 65-68C. Dextran sulfate may be used to
enhance the rate of reassociation. After hybridization, the
filters are washed extensively for remove background
(unhybridized) probes. The washing procedure is carried out
at elevated temperature and reduced salt concentrations to
remove non-specific DNA/DNA hybrids as well.
Preparation of Probe DNAs
As mentioned previously, the probe DNAs represent
25 variable DNA fragments, chosen on the basis that they
represent one-allele of a polymorphic genetic region. In
this context the polymorphism is one of length. The
variability in fragment length is a result of a difference in
the number and/or location of endonuclease restriction sites
3o which were attacked during the generation of the fragments.
Thus, if all individuals possessed a DNA fragment of similar
~lS3~4
l si~e which hybridized to the probe DNA, the region would be
considered monomorphic and of little utility with respect to
the subject invention. Whereas when individuals possess DNA
fragments of dif~erent sizes which hybridize with the probe
5 DNA fragment; then that fragment can be said to represent an
allele of a genetic region which displays size polymorphism.
The evaluation of probes is then of critical importance and
may be considered to consist of the two interrelated steps of
probe generation and probe identification.
lO Probe Generation
The generation of probes may ~e accomplished
accordin~ to art recognized procedures for the construction
o~ a collection of cloned DNA fragments The steps normally
include: digesting a DNA sample with a specific endonuclease,
recovering ~ract~ons of D~A of appropriate size ~rom the
digest, precipitating the fragments, introducing the
fragments in to an appropriate cloning vector, transforming a
competent host organism with the vector, and recovering
colonies containing the cloned probe DNA. A variety of
endonucleoases and vectors exist which may ~e used in the
generation of probes. The methods f~r accomplishing the
cloning is well known in the art (see for example, Molecular
_loning: A Laboratory Manual, T. Maniatis, et al., Cold
Spring Harbor Lab 1982). Two human DNA probes generated in
such a manner are pAW 101 and pLM 0.8. Samples of E. coli
harboring pAW 101 and pLM 0.8 were deposited with The
American Type Culture Collection, 12301 Parklawn Drive,
Rockvi]le, Maryland on February 8, 1984 where they were
assigned the accession numbers ATCC 39605 and ATCC 39604,
respectively and the requisite fees were paid. Access to the
cultures will be available during the pendency of the patent
l application to one determined by the Commissioner to be
entitled thereto under 27 C.F.R. 1.14 and 35 U.S.C. 122.
All restrictions on availability of said culture to the
public will be irrevocably removed upon the granting of the
5 instant application and said culture will remain permanently
available during the term of said patent. Should the culture
become nonviable or be inadver-tently destroyed, it will be
replaced with viable culture(s) of the same taxonomic
description.
Alternatively, cDNA probes may also be employed.
These probes are generated initially from RNA by a reverse
copying procedure and is detailed in Example II herein or EP0
Publication No. 0 0~4 796.
Irrespective of the method used to generate the
15 probes, once obtained, each probe must be evaluated for
usefulness in the testing procedure.
Identification of Useful Probes
To evaluate the efficacy of a particular probe from
the collection of probes generated above, DNA is isolated
from four different individuals and separately digested with
a restriction endonuclease. These DNAs are subjected to
agarose gel electrophoresis, running a mixture of three of
the individuals' DNAs in one lane and a sample from the
fourth individual in an adjacent second lane. The
electrophoresed DNAs are blotted as described previously.
Single-stranded DNA is isolated from an individual clone
selected from the group of potential probes containing clones
generated above. The probe DNA is labelled and used to
hybridize with the electrophoresed DNA of the four
individuals. If the tested probe yields more bands in the
lane with the three lndividuals' DNAs than in the lane with
-14-
1:~15;~4
l the one individual's D~A, it becomes a candidate to detect
polymorphisms. Probes identified by the foregoing procedure
are further tested by hybridization against a sufficiently
large population of test individuals to effectively determine
5 the extent of polymorphism. Probes corresponding to regions
with at least four different alleles present in the
population with frequencies greater than 10% ~ach are
incorporated into the test.
According to a preferred embodiment of the
10 invention, a collection of polymorphic probes are employed
rather than the reliance on a single polymorphic probe. This
use of multipIe probes increases the sensitivity of the test
dramatically. ~or example, if ten different probes are
employed and each probe identifies a polymorphic region
consisting of eight equally frequent occurring alleles,
approximately a million individuals could be uniquely
identified.
The parameters to be evaluated when selecting a
particular probe for inclusion in the collection comprise the
20 degree of polymorphism, that is, the number of alleles and
the frequencies that the alleles are present in the
population to be tested. The mere existence of a large
number of alleles, e.g., 60, at a particular probe locus in
and of itself would not ensure a useful probe if, for
example, 99.9% of the population to be tested possessed one
allele and the remaining 0.1% was distributed among the other
59 alleles. Thus, the frequency of occurrence of the various
alleles is an important consideration.
The number of individual probes in a probe set
3o could be quite large, 100 or more, practical limitations
would restrict the number to from 1 to about 40, more
preferably from 1 to about 20.
1~53~;?4
1 The number of alleles per polymorphoric genetic
locus can be large, from 2 to about 60 or more, but more
preferably from 2 to about 40. Optimally, the alleles will
occur in roughly equally frequency.
5 Autoradiography
The hybrid is visualized by means of
autoradiography. Prior to the hybridization, the probe DNAs
are labelled with a radioactive isotope, usually 32p. The
specific activity of approximate 108 counts per min per ug
10 of DNA is required and normally involves labelling with at
least two labelled nucleotides (TTP and dCTP 400 Ci/mmol).
The radioactive hybridized probe is localized using art
recognized procedures involving exposure of film to the
radioactive emissions. The radioactive hybrids essentially
15 take their own picture hence the term autoradiography.
Although autoradiography is an art recognized
procedure for the localization o~ the h~brid molecules, the
invention is not restricted to this particular mode of analy-
sis. The hybrids of interest may be detected by means of any
suitable analytically detectable reagent. For example,
fluorescent detection, colorimetric reactions, immunological
reactions, or enzymes or other protein-labelled reagents are
also useful in the detection of the hybridized probes.
3o
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12153(~4
1 ~XAMPLE I
This example illustrates the isolation of DNA from
human peripheral blood. DNA so isolated is useful in the
evaluation of probe DNA.
Ten to twenty cc. of peripheral blood is collected
using EDTA as anticoagulent (~lood may be processed
immediately or frozen at 70C).
The blood is transferred to a 50 ml tube and an
equal volume of lysing buf~er ( 1 mM MgC12; 1 mM
lO NaH2PO4, pH 6-~: 0.8% Nonidet P-40; 0.4% deoxycholic
acid, sodium salt) is added. The tube is inverted 25-50
times to mix well.
The mixture is transferred to a 50 ml plastic
Sorvall tube and spun in an SW 34 rotor at 10,000 rpm
(12,000 g) for 30 minutes. The supernatant is disçarded and
the pellet is suspended in 10 ml of TNE (10 mM Tris, p~ 8.3;
150 mM NaCl; 5 mM EDTA). The pellet is disrupted by shaking
the tube violently. 1.5 ml of 10% SDS ~f.c. 1.0~) is added
and inverted several times. Three ml of 5 M NaClO4 (f.c.
1.0 M) is added and mixed. An equal volume of chloroform:
isoamyl alcohol (24:1) is then added and the tube is placed
on a New ~runswick gyrotory shaker at 3500 rpm for 15
minutes. The phases are separated by a 10 minute spin at
3,000 rpm in Damon centrifuge.
The aqueous (top) phase is removed with an inverted
lO ml pipette, without cotton (a siliconized Pasteur pipette
may also be used) and transferred to a fresh 50 ml tube. The
organic (bottom) phase, is discarded , an equal volume of
Chl:IAA (24:1) is added and extracted and separated as
3O before. The extraction is repeated until the interphase
after phase separation is clear. This usually requires 3-5
extractions.
lZ~53~)4
lThe aqueous phase from the final extraction is
transferred to a plastic beaker. Two to two and one half
volumes of -20C 95% EtOH is added by slow pouring down the
- side producing two phases; aqueous-DNA phase on bottom and
5 EtOH on top. A clean, dry glass rod is wound in this
solution until the tw~ phases mixed. The DNA precipitates at
the aqueous-~t~H interface and is collected on the rod.
After the two phases have mixed, the rod is removed and air
dried for 10 minutes.
lOThe rod is placed in 15 ml tube and covered with
parafilm. Three holes are punched into the parafilm with an
18 gauge needle and the sample is dessicated 20 minutes.
The parafilm is removed and 0.5-1.0 ml of 0.01X SSE ~1.5 mM
NaCl; 0.15 mM EDTA, pH 7.0), is added. The sample is capped
15 and suspended overnight at 4C on an Ames rocker.
The amount of DNA in suspension is determined by
recording the O.D. of a l/20 dilution of sample. 25~ul of
the DNA suspension is added to 475 ,ul of distilled water,
transferred to a cuvette and the O.D. recorded at 260, 270
and 280 using a cuvette filled with 0.01X SSE to zero each
reading. The concentration of DNA in the suspension in mg/ml
(~g/,ul) equals the reading of a l/20 dilution at O.D. 260
because the O.D. 260 of 1.000 = 50 ~g/ml. A dilution is made
3o
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12153Q4
1 to keep the O.D. 260 between 0.100 and 1.000 where the
correlation between DNA concentration and O.D. is linear.
O.D. readings above 1.500 are not accurate. The ~.D. 260/280
should be 1.8 or greater and measures the amount of protein
5 contamination. For example the following O.D. values were
recorded from 0.5 ml of a 1/20 dilution of a DNA suspension
form peripheral blood:
260 270 280 260 260 concentration
270 280
lO 0.350 0.280 0.190 1.25 1.84 0.35 ~g/~l
0.350 x 50 ~g/ml x 20 = 350 ~g/ml = 0.35 ~g/~l
3o
--19--
1S3~
1 EXAMPLE II
This example illus-trates a method for the
generation of human DNA probes.
A. Messenger RNA Isolation
1. Between 10 _108 human cells are suspended
in 2 mls ice-cold Rin~er's and centrifuged at 2000X g for
5 minutes at 4C.
2. Following aspiration of the supernatant, the
cells are resuspended in ice-cold lysis buffer. The buffer
10 being comprised of
0.14 M NaCl
1.5 mM MgC12
10 mM Tris-Cl pH 8.6
0.5~ NP~40
~,OOG units/ml RNasin (Biotec)
3. The suspension is vortexed for 10 sec. then
underlayed with an equal volume lysis buffer containing
sucrose (24% w/v) and 1~ NP-40 and stored on ice for 5
minutes.
4. The suspension is centrifuged at lO,OOOX g for
20 minutes at 4C in a swinging-bucket rotor.
5. The turbid; upper (cytoplasmic) layer is
recovered and an equal volume of 2X PK buffer is added.
2XPK buffer: 0.2 M Tris-Cl pH 7.5
25 mM EDTA
0.3 M NaC1
2~ S.D.S.
Followed by the addition of proteinase K at a final
concentration of 200)ug/ml and incubation at 37C for 30
3o minutes.
-20
121S3~4
l 6. The layer is then extracted once with
phenol/chloroform and the aqueous layer recovered, to which
is added 2.5 volumes of ethanol and stored at -20C for at
- least 2 hours.
7. The fraction is centrifuged for 10 minutes at
5000X g at 0C and the resulting pellet washed with 75%
ethanol containing 0.1 M sodium acetate.
8. The nucleic acids are redissolved in a small
volume (-50,ul) of:
S0 mM Tris-Cl pH 7.5
1 mM EDTA
9. To the resuspenaea iraction is added MgCl2 to
a final concentration o~ lOmM and R~asin ~Biotec) to 200~
units Iml . The suspension is then incubated for 30 minutes at
37C.
lO. Following incubation, EDTA and SDS are added
to a final concentration of lOmM and 0.2%; respectively.
11. The suspension is extracted with
phenol/chloroform and Na acetate pH 5.2 is added to 0.3 M and
the nucleic acids are precipitated with 2 volumes of ethanol.
12. The RNA in 70% ethanol is stored at -70C.
B. Selection of poly A RNA
1. Oligo ~dT)-cellulose is equilibrated in
sterile 2x loading buffer. The buffer is composed of 40 mM
Tris-Cl pH 7.6.
1.0 M NaCl
2 mM EDTA
0.2% SDS
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1 2. The oligo- (dT) -cellulose is used to form a 1 ml
column and washed with 3 column volumes each of:
a) sterile water
b) 0.1 M NaOH/5mrl EDTA
c) sterile water
3. The effluent pH should be less than p~ 8Ø
4. The column i6 then washed with 5 volumes of
loadi~g ~uf~er.
5. R~A isolated in step A is dissolved in sterile
10 H2O and heated to 65C for 5 minutes. An equal volume of
2x loading buffer is then added and the sample i5 cooled to
room temperature ~25C),
6, The sample is then app~ie~ to the column and
the flow-through is collected. The flow-through is then
15 heated to 65C, cooled and reapplied to the column.
7. The column is then washed with 5-10 volumes of
loading buffer, followed by 4 volumes of loading buffer
containing 0.1 M NaCl.
8. Fractions are collected and read at 0D26o.
Initial fraction will contain poly(A) RNA in high
concentration while later fractions will have little or no
OD260 absorbing material.
9. The poly(A) RNA is eluted from the column
with 2-3 volumes of sterile:
10 mM Tris-Cl pH 7.S
1 m~l EDTA
0.05~ S.D.S.
10. Na Acetate (3 M pH 5.2) is added to the eluant
to a final concentration of 0.3 M and 2.2 volumes ethanol are
3o then added.
11. The RNA is centrifuged at 0C at 5000X g for
10 minutes.
3~
-22-
;3~)4
l 12. The pelelt is redissolved in water.
(10 cells yields 1-5 ug p31y (A) RNA)
C. Synthesis of the First DNA Strand
1. The reaction conditions below assumes a
5 starting amount of 50 ~g of polyA mRNA. For amounts
greater or less than 50 g the reaction mixture may be scaled
proportionately.
2. Reaction mixture comprising:
Final
10Rea~nt Amount to AddConcentration
lO mM dATP 25 ~l 500 ~M
10 mM dGTP 25 ~l 500 ~M
10 mM dTTP 25 ~l 500 ~M
15 2 mM dCTp 25 ~l lO0 uM
5 X Reverse Trans-
criptase buffer
250 mM Tris 8.2; 50 mM Tris
250 mM KCl; 30 mM 50 mM KCl;
6 mM MgCl2
MgC12 100 pl
200 mM DTT 25 ~l 10 mM
Poly(A) mRNA (50 ~g)
RNasin (Biotec)
25 placental RNase
inhibitor 5 pl
Avian myleoblastosis
virus reverse
transcriptase 20 ~l 300 ~/ml
3o Oligo(dT) 12-18
600 ug/ml 37.5 ul 45 ~g/ml
32p_dcTp l-lO uCi/500 ~l
reaction
distilled H2O To final volume: 500 ~l
1 3. The reaction is performed in a 1.5 m]
siliconized Eppendorf tube and initiated by the addition of
the mRNA.
4. The reaction mixture is incubated at 42C for
5 60 minutes, then 10 ul of 500 mM EDTA is added to stop the
reaction.
5. 1 ~l of the reaction mix is precipitated with
T.C.A. and counted to determine the efficiency of 1st
stranded synthesis. Generally, 17-25~ e~ficiency is
lO obtained, rarely as high as 40%.
6. ~0 ~ Ci of 3 P-dCTP/500 ul will yield a
specific activity of 2.2x106 cpm/~g of single stranded
DNA. The specific activity allows maintaining of the product
in subsequent step without wasting too much o~ the cDNA.
7. The sample is extracted twice with an equal
volume of phenol saturated with 50 mM Tris pH 8Ø
~ . The phenol is extracted twice with ~Jolumes of
ether. After which is added 3 M Na acetate to 0.3 M.
9. Three volumes of 95~ ethanol are added and the
20 mixture is placed on dry ice-ethanol for 5-10 minutes then
warmed to room temperature.
10. The mixture is spun in a microfuge ~or lS
minutes after which time the supernatant is discarded and the
pellet washed with 75~ ethanol.
ll. The DNA is redissolved in 0.5 ml of 300 mM Na
acetate and steps 9 and 10 are repeated.
12. The DNA is resuspended in 200,ul of distilled
water, layered on 5-20~ alkaline sucrose gradient (30 mM
NaOH, 2 mM EDTA) and spun for 40 minutes in an SW-40 rotor at
3o 37,000 rpm at 4C.
-24-
~Z~S304
l 13. 0.5 ml fractions are collected from the top of
the gradient and place into 25 ~1 1 M Tris pH 6.8 and each
fraction counted.
14. Five thousand-ten thousand counts per minute
5 from every other fraction are removed and run on an alkaline
agarose gel. This permits a size distribution estimate to be
made. Generally fractions which have cDNA of less than 500
nucleotides are discarded. Fractions particularly useful
(i.e., at least 500 nulceotides long) usually occur at
10 fraction 12 from the bottom of the tube. Therefore while the
gel is running and developing, fractions 1-10 lincluding the
pellet) are pooled and dialyzed against 2 liters of water
fractions 11, 12, 13 and 14 are also dialyzed but
individually, The gel pattern will indicate whether or not
further pooling is necessary. In general material greater
than 500 nucleotides will account for 60% of the
TCA-precipitable counts.
15. The ss cDNA is then concentrated with
sec-butanol to a volume of ~400 ~ul followed by extraction of
the butanol with ether.
16. To the extract is added 40 ul 3 M Na acetate
and the remainder of the tube is filled with 95~ ethanol.
Precipitate on ethanol-dry ice for 5 minutes then place the
tube in a water filled bucket of SW-27 rotor and centrifuge
at 25,000 rpm for 60 minutes.
17. The ethanol is decanted and counted. The
ethanol should contain less than 1% of the counts. Wash the
pellet with ethanol and count the wash solution again; less
then 1% of the counts should be removed. All counts should
3o remain in the pellet which is lyphilized for 10-20 minutes
and then resuspended in 100 jul of water.
1;~1S~4
l D. Second Strand Synthesis With Klenow
1. The reaction mix below is for a 1 ml reaction
at a concentration of ss cDNA of 2-5 ~g/ml.
Final
Reagent Amount to Add Concentration
10 mM dATP, TTP, CTP, GTP 50 ,ul 500 pM
700 mM KCl 100 ~l 70 mM
5 mM mercaptoethanol100 ~l 0.5 mM
(~dd 1.8 ul of stock
Eastman ~14 M) t~ 5 m~
H2~ to yield 5 mM)
10 x Xlenow buffer 100 ~ 30 mM Tns
300 mM Tris pH 7.5 4 mM MgCl2
40 mM MgCl2
Klenow polymerase
Boehringer-Mannheim 150-200 units/ml
SS c DNA 2.5 ~g
distilled H2O To final volume
of 1000 ~1
2. The reaction is incubated at 18-20C for 5-6
hours.
3. The mixture is extracted twice with phenol-Tris
25 pH 8 and ether.
4. An aliquot (2-10,000 cpm) is saved for gel
analysis.
5. The remaining extract is dialyzed over night
against water in a colloidon bag.
3o E. S1 Reaction
~1S;~04
l 1. The volume of the Klenow reaction of step D
will increase to 5-6 ml during dialysis. The volume is
adjusted to the next highest ml with d H20 and one-tenth
volume of 10 x Sl buffer is added:
3 M NaCl
0.3 M Na Acetate pH 4.5
100 mM ZnCl2
2. S1 nuclease (Sigma) is added to a final
concentration of 10 units/ml and incubated at 37C for 30
lO minutes and stop the reaction by the addition of 500 mM EDTA
to a final concentration of 100 mM. An aliquot is saved for
gel analysis.
3. The reaction mix is extracted twice with phenol
and twice with ether. The extract is then dialyzed for 5-6
15 hours at room temperature vs water with at least one change
of water and then concentrated with sec-butanol to ~400 ,ul.
4. The sample is loaded onto a neutral 5-20%
sucrose gradient (0.1 M NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA)
and centrifu~ed at 37,000 rpm is ~W-40 rotor for 20 hours at
4C.
5. Fractions of 0.5 ml are collected from the top
of the tube. Fractions 1-14 will contain ~500 bps of ds
cDNA. Gels are run to verify the size distribution.
6. The fractions are dialyzed exhaustively
overnight against distilled water.
7. The sample is concentrated to ~400 ~ul with
sec-butanol and precipitated with Na acetate and ethanol
twice. The pellet is washed each time with 75~ ethanol. The
DNA must be contaminant free.
8. The ds cDNA is then lyophilized.
F Tailing Reaction
.
1. The reaction conditions below are for 1 ~g ds
3~4
l cDNA and may be scaled up or down accordingly.
Stock solutions: 50~uM dCTP 10 m~l CoCl2
2X cacodylate buffer: 250 )ul 1.2 M Na-cacodylate, pH 7.19
with HCl
250 ~l 1 mM DDT
750~ul H2O
Reagent Amount to add
lO 2X cacodylate buffer 200 ~l
cDNA (50 ng/ul) 20 ~l (lug)
50 ~M dCTP 40 ~l
20 ~M CoC12* 20 ~l
25 mg/ml BRL nuclease free BSA 8 ,ul
15 dH2O 68 ~l
TdT IBethesda Res. Lab) 44 ~1 (760 u/ml final conc)
* Add CoC12 just before BSA ~r it will precipitate,
2. The reaction mixture (-TdT) is incu~ated at
20C for 20 minutes.
203. The TdT is then added and the incubation
continued for another 20 minutes.
4. The reaction is stopped by the addition of 8)ul
of 500 mM EDTA and then extracted twice each with phenol then
ether.
255. The sample is precipitated as above with sodium
acetate, ethanol in the S~-27 rotor.
6. The pellet is washed with 7.5~ ethanol,
lyophilized and resuspended in 50~ul of distilled water.
3o
-2~-
lZ~S~Q4
1 G. A ealing Tailed cDNA to plasmid-dG
1. The annealing reaction is performed in 10 ~1
sealed capillary tubes.
2. The reaction mix comprised:
ds cDNA 1 ~ul (5 ng)
plasmid 1 ~1 (20 ng)
lOX annealing buffer
1 M NaC1
100 mM Tris; pH 7.5
10 mM EDTA
distilled H20 7 ~ul
3. The mixture is incubated at 68 for 8 min~tes,
then at 42~C for 2 hours after which time the water bath is
turned off and the reaction mix allowed to equilibrate to
room temperature (5 hours - overnight).
3o
-29-
lZlS3~4
1 EXAMPLE III
This example illustrates the methods of
identification of probes which are useful in the detection of
polymorphisms in humans.
1. DNA is isolated from the peripheral blood of 4
different human subjects as described in Example I.
2. The four samples of DNA are restricted
separately with restriction enzyme EcoRI according to the
following procedure.
a) The following components are added to a 1.5 ml
eppendorf tube:
(1) Enough of the DNA solution for lO~ug
(usually between '10 ~l and 50 ,ul).
(2) Distilled water, if necessary, to adjust
to ~he final reaction volume.
(3) The appropriate amount of the specific 5X
endonuclease digestion buffer made to the
manufacturer's recommendations.
(4) Restriction endonuclease in 1.5 to 2.5
fold excess, i.e., 15 units to 25 units
for a lO~ug digestion.
b) The mixture is vortexed 1-2 seconds or the tube
is flicked with a finger several times to mix.
c) The mixture is spun in eppendorf micro-
centrifuge 10-15 seconds to pellet reactants.
d) The pellet is incubated 2-16 hours at 37C.
e~ The reaction is stopped to store for future
electrophoresis adding:
(1) 1/10 volume of 0.1 M EDTA, pH 7.0;
f.c. 10 mM
(2) 1/10 volume of 5% SLS; f.c. 0.5%.
~Q~
1 (3) 1/10 volume of 3 M NaCl or 3 M NaAcetate;
f.c. 0.3 M
(4) 2 to 2~ volumes of cold 95~ EtOH;
f,c. about 70%
The sample may be stored to -20C for up to several
months.
Samples can be precipitated quickly by placing an
eppendorf tube containing the digested DNA, stop reactants,
and EtOH in a dry ice-EtOH bath for 2-5 minutes depending on
10 the volume until the EtOH is viscous. The samples should not
be frozen. The sample is spun in microfuge to pellet.
f) To stop reaction which is to be loaded to gel
immediately after digestion, add SX ficoll marker dye
solution to a final concentration of lX. This is done with
samples where the final volumes is less than 75 jul.
g) A typical reaction mixture is constructed as
follows:
10mgDNA H 0 5x buffer EcoRI 0.lM 5% 3M 95
--2-
5~/,ul EDTA SDS NaCl EtOH
2020~ul 16~1 10~1 4~1 6.25iul 6.25,ul 7.0,ul 14.0~1
and incubated at 37C for 2 hours. The EDTA, SDS, NaCl, and
EtOH are added as indicated and store at -20C or add 12.5 ~1
of 5X Ficoll marker dye and load on gel.
3. The DNAs are subjected to electrophoresis as
described in Example II running 5 ,ug of each of three of the
individuals DNAs in one lane and 5 ~lg of DNA from the fourth
individual in an adjacent lane.
4. The electrophoresed DNAs are then blotted
according to the following procedure:
3o
~lS3~4
l a) The D~A is denatured in the gel by transferring
the gel to a blotting bowl (round pyrex, 190x100 mm)
containing 250 ml of 1 ~I KOH, 0.5 M NaC1 and shaken at 200
rpm at room temperature on a New Brunswick gyrotory shaker 25
minutes for an 0.8~ gel to 30 minutes for a 1.2% gel.
b) Precut nitrocellulose sheets (9~2x15 cm) are
placed in 200-300 ml of distilled water to thoroughly wet.
c) The solution is decanted and saved (KOH-NaCl
solution may be used to denature up to 10 gels.) The gel is
10 rinsed with distilled water (200-300 ml). All rinsed water
is removed with Pasteur pipette. 250-300 ml of 1 M Tris, pH
7.0 is added and shakein~ is continued at room temperature at
50 rpm for 35 minutes.
d) The gel is neutralized by decanting and add
250-300 ml of 1 M Tris, plI 7.0 and continuing to shake for 30
minutes. The Tris solutions are sa~ed and adjusted back to
pH 7.0 with concentrated HC~ up to 10 times.
e) Optibnally, the gel is decanted, 250-300 ml of
1 M Tris, pH 7.0 is added and shaking is continued for 25
20 minuteS~
f) All of the Tris is decanted and removed with a
Pasteur pipette. The gel is equilibrated by adding 250-300
ml of 6SSC (lX = 0.15 M NaCl, 0.015 M NaCitrate) and shaken
for 20 minutes.
g) The distilled water is decanted from the
nitrocellulose and 100-200 ml of 6X SSC is added.
h) Using a pyrex 28X18X4 cm tray add 600-700 ml of
6X SSC. A wick of two strips of Whatman 3 M (15~zx38 cm) is
wetted in the 6X SSC solution. A plastic blotting platform
~18~xl9xl cm) is placed in the middle of the tray and the
Whatman 3 M wick is centered on the platform so that each end
is submerged in the 6X SSC solution.
~21S31~4
1 i) While wearing gloves, the gel is transferred
from the bowl to the wick. The gel is rubbed with gloved
fingers to ensure contact with gel and wick.
j) The presoa~ed nitrocellulose ISchleicher and
5 Schuell, Keene, N.H.) is placed on the gel and positioned
over the lanes to be blotted. Rubbing with gloved fingers
ensures contact of the gel and nitrocellulose and the
appearance of no air bubbles. The gel not to be blotted is
trimmed and discarded. Three pieces of a 1 ml pipette are
10 placed along each side of the gel to avoid short circuits.
One piece of Whatman 3 M (15~x9~) is wetted and placed on top
of nitrocellulose. Another similar sized dry piece of
Whatman 3 M is added. About 10 cm of lO~x12 cm brown towels
(No. 237 Singlefold Garland Sof-~nit Towels; Fort Howard
15 Paper Company, Green Bay, Wis. 54305) are stacked on top of
gel. Cover by plastic wrap pulling tight around tray. The
apparatus is left for 12 20 hours at room temperature. The
blotting platform is placed on top for weight.
k) The towels are removed (some of the top ones
20 may still be dry) along with two pieces of Whatman 3 M
exposing nitrocellulsoe paper. A new razor blade is used to
cut the nitrocellulose sheet into three strips containing 2
or 3 lanes worth of DNA (2 lanes each with the 8-lane well
former and 3 lanes each with the 10-lane well former). The
lower left corner of each strip is nicked for orientation and
one, two or three holes are punched into the bottom of the
appropriate strips for identification. After the strips have
dried, they can be labelled with a marking pen.
3o
-33-
12153Q4
l l) The strips are placed in 250 ml of 2X SSC in a
blotting tray. Each side of the strips are rubbed with
gloved fingers to remove bits of aragose. The strips are
placed on Whatman No. 1 filter paper to air dry for 10-20
5 minutes. The strips are then placed between two pieces of
Whatman 3 M paper and wrapped in aluminum foil. The outside
is labeled with marking pen and may be placed in vacuum in
dessicator for up to 6 months.
5, E. coli MC1061 carrying recombinant plasmids
lO are cultured in 100 ml L broth from an individual colony of
the library generated in Example II and plasmid DNA is
isolated according to the following procedure:
a) The cells are centrifuged at 5000 rpm for 5
minutes at 0C.
b) The cells are washed with ~ volume of TE (10 mM
Tris-HCl, 1 m EDTA pH8) at 0C,
c) The cells are resuspended in 3 ml of 25%
sucrose, 0.05 M Tris HCl pH 7.5 at 0C and 0.3 ml lysozyme
(10 mg/ml in 0.25 M Tris-HCl pH 7.5) is added; followed by
incubation on ice for 5 minutes with occasional gentle
swirling.
d) 1.2 ml of 250 mM EDTA pH8 is added and
incubation on ice is continued for 5 minutes.
e) 48 ml of Triton solution:
2 ml 10~ Triton X 100 (Sigma)
50 ml 250 mM EDTA pH:
135 ml H2O
is added and incubated on ice for an additional 10 minutes.
f) The mixture is spun for 30 minutes at 25,000
rpm at 0C.
lZlS3l~4
l g) The supernatant is re~o~ed and the volume is
adjusted to 8.7 ml followed by the addition of 8.3 g of CsCl
and 0.9 ml of 10 mg/ml ethidium bromide (Sigma #E-8151). The
refractive index should be between 1.390 and 1.396.
h) The sample is centrifuged at 35-38K at 20C for
48-72 hours and visualize the bands by illuminating the tube
with long wavelength ultraviolet light.
i) The lower band which contains the supercoiled
DNA is collected by side puncture of the tube with a 21 guage
10 needle.
6. The pAT 153-human DNA recombinants are labelled
with 3 P by nick translation as is well known in the art
('IA Manual for Genetic Engineering. Advanced Bacterial
Genetics" by Davis, R. W., Botstein, D. and Roth, J.R. 19~0
15 Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.
168-170).
al 20 ,ul of water minus the volume of the DNA
solution which is to be added is placed in a microfuge tube.
b) 2.5 ,ul of 0.5 M Tris pH 7.5, 0.1 M MgS04,
10 mM pTT, 500 ~g/ml BSA is then added.
c) 2.5 ul of a solution containing 0.2 mM each
dNTP followed by the addition of 100 mg of pAT 153 human
recombinant DNA from step 5 above.
d) A DNase stock solution:
DNase 1 mg/ml in
50 mM Tris pH 7.5
10 mM MgS04
1 mM DTT
and 50% glycerol
3o i5 previously prepared and stored at -20C.
15;~(~4
l e) The DNase stock solution from (d) abo~e is
diluted at 0C l/40,000 into ~0 mM Tris pH 7.5, 10 mM MgSO4
1 mM DTT and 50 ~g/ml BSA and 0.5 ul of the diluted DNase is
added to the reaction mixture.
f) 10 ~Ci of each 3 P dNTP in aqueous solution
is added.
g) The entire reaction is initiated by the
addition of 0.1 ~l of 2 mg/gl E. coli DNA polymerase I and
incubated at 14C for 3 hours.
h) 25 ~l of 0.02 M Na3 EDTA 2 mg/ml carrier DNA,
and 0.2% SDS is added to stop the reaction.
i) The reaction mixture is loaded onto a 0.7x20 cm
Sephadex G-50 (medium) column preequilibrated with 10 ~I Tris
Na3 EDTA at pH 7.5 (TE) and washed with same.
i) 0.5 ml effluent samples are collected in
polypropylene tubes. The DNA appearing after 2 ml of wash.
The location of the 32P-labelled DNA is followed with a
hand-monitor and the first peak is collected ignoring the
tail.
7. The labelled probe from step 6 is hybridized to
the blotted genomic DNAs from step 4 according to the
following procedure:
a) 300 ml of prehybridization solution is prepared
as follows:
1) 100 ml 3X PO4 (0.75 M Na2PO4, 0.75 M
NaH2PO4, 0.01 M NaPyrophosphate ~
2) 90 ml 20X SSC (3 M NaCl, 0.3 M NaCitrate)
3) 92 ml distilled water
4) 15 ml 0.5% BFP (0.5 g per 100 ml each of;
3o bovine serum albumin, ficoll, and polyvinylpyrolidone-360)
12~4
1 5) 3 ml 5~ g/~ 1 ssDNA (denatured salmon sperm
DNA), The solution is transferred to plastic bin with top
(20x14~xlO~ cm) and heat to 68C in water bath. The filters
to be hybridized are added and incubated for 4 to 6 hours at
5 68C
b) For hyhridization strips, 3 to 4 strips are
wrapped around a siliconized glass vial and inserted into a
plastic scintillation vial containing 2 ml of hybridization
solution. For hybridization of nitrocellulose sheets in
lO hags, the appropriate amount of hybridization solution is
added and the bag sealed with heat from a Sears seal-it
device,
The hybridization solution is made as follows for
hybridization in a vial:
(1) 80 ~1 o~% BFP
(2) 20 ~1 0.1 M EDTA, pH 7.0
(3) 20 ~1 10~ SDS
(4) 20 ~1 5 ~g/~l ssDNA
~5) varia~le 3 P nick translated pro~e to give
2-4x106 counts/ml
(6) variable distilled water to adjust to l900jul
boil 12 minutes; ice 7 minutes
l7) lOOjul 20X SSC
2000,ul
c) Wearing gloves, the strips are removed directly
from the prehybridization solution and the appropriate 3 or 4
strips are wrapped around a siliconized glass vial (19x48 mm
with cap) and inserted into a plastic scintillation vial (28
mm diameter) containing the prepared hybridization solution.
3o
3~
-37-
12153~4
1 Parafilm is wrapped aro~nd the canned lid. Tapping
several times ensures the filters are all at the bottom of
vial. The filters are incubated 20-24 hours at 68C in a New
Brunswick gyratory water bath with slow shaking (setting
5 number 3). Note: If there are less than 3 strips to wrap
around the vial, one or two blank strips which have been
prehybridized can be added.
d) The filters are washed in 2X SSC, 0.5% SDS as
follows: 6-9 liters of wash solution is prepared depending
lO on the number of filters to be washed. To a glass carboy
with stopcock at the bottom is added:
(1~ 600 to 900 ml 20X SSC
(2) 300 to 4~0 ml 10% SDS
(3) 5100 to 7650 ml distilled water
15 A stir bar is placed at the bottom and a thermometer is
suspended from the top. The solution is heated to 68C on
hot plate with stirring.
1 to 1~ liters of wash solution is collected in
plastic bin. The filters are removed after hybridization
(wearing gloves) and immersed immediately in wash solution.
Millipore forceps are used to unroll and transfer filters.
e) The filters are transferred to 1 to 1~ liters
of fresh wash solution and incubate 7-12 minutes at 68C in
water bath. The first wash solution is carefully discarded
down the drain with plenty of water to flush.
f) The filters are again transferred to 1 to 1~
liters of fresh wash solution. Continue to transfer every
7-12 minutes and incubate at 68C until all of the wash
solution is used (4-7 washes).
3o
-38-
~2:1~;304
l g) The final transfer is to 1 liter of wash
solution containing 0.1X SSC, 0.5~ SDS (945 ml distilled
water, 50 ml 10% SDS, 5 ml 20X SSC) heated to 68C.
Incubation is at 68C for 10 minutes.
h) The filters are removed and rinsed in 500 ml of
2X SSC at room temperature. Filters are placed on sheet of
Whatman No. 1 to air dry 15-30 minutes.
i) The 6 strips from two gels are taped on yellow
paper from an x-ray film pack, labeled, covered with plastic
10 wrap and placed in cassette with built in intensifying
screens. In dark room, the cassette is loaded with 8x10 inch
X-Omat AR x-ray ~ilm placing film ~etween nitrocellulose
strips and screen. The cassette is closed and placed in a
freezer at -70C,
j) The x-ray film is de~eloped in 24 to 48 hours.
The film is removed from cassette and developed in dar~ room
with yellow safe light on. The cassette may be reloaded if
another exposure is required.
8. If the tested probe yields more bands in the
lane with three individuals' DNAs than in the lane with only
one individual's DNA it becomes a candidate to detect
polymorphisms.
9. Probes identified in step 8 are further tested
by hybridizing them against a larger series of human DNAs to
determine the extent to which the cloned region is
polymorphic. Probes corresponding to regions with at least
four different alleles present in the population with
frequencies greater than 10~ each are incorporated into the
test for paternity or the test for individual identity.
3o
-39-
12153~)4
1 EXAMPLE IV
This illustrates the performance and evaluation of
a paternity test employing the subject invention.
1. Blood samples are taken from the mother, child,
5 and putative father and DNA purified as described in Example
I.
~ . These DWAs separately reacted with restriction
enzyme EcoRl as described in Example III.
3. These DNAs are subjected to electrophoresis as
10 described in Example II running 5 ~g of each of the mother's
and the putative father's DNAs in one lane and 5~ g of DNA
from each of the three individuals in an adjacent lane.
4. The electrophoresed DNAs are blotted as
described in Example III.
5. The set of "paternity probe" DNAs is
lahelled with 32p as described in Example III.
6. The labelled probe DNAs from step S are
hybridized with the blotted genomic DNAs from step 4 as
descri~ed in Example III.
: 20 All genes of the child will be derived from either
the mother or father. Therefore, if the putative father is
the biologic father all bands present in the lane with the
child's DNA will also be present in the lane w~thout the
child's DNA. Conversely, if the putative father is not the
25 biologic father, new bands will appear in the lane with the
child's DNA.
3o
-40-
l~S304
1 EXAMPLE V
This example provides specific techniques for and
evaluation of a paternity test.
A. DNA PURIFICATION FROM BLOOD
1. Samples of blood (S to 10 ml) should be
collected in tubes containing EDTA or Citrate as
anticoagulant and stored at 4 C until processed.
2. Resuspend cells by inversion and centrifuge at
2,000 rpm for 10 minutes at 4 C. Remove serum ttop) without
10 disturbing buffy coat.
3. Add e~ual volume of blood lysis buffer ~0.32
sucrose, lOmM Tris pH 7.6, 5mM MgCl, 1~ ~riton X-100) at
4 C and mix well by inversion. Transfer into a 50 ml
polypropylene conical tube (e.g. Corning, Falcon), rinse
15 blood tube and adjust final volume to 4 times the original
blood volume. Mix well and centrifuge at 2,000 rpm for 10
min. at 4 C.
4. Decant supernatant. If pellet is not clean
(i.e. too much red cell contamination), then resuspend pellet
in 3 ml of lysis buffer and centrifuge again.
5. Resuspend whitish-pink pellet in 2.5 to 5 ml of
DNA lysis buffer (10 mM Tris pH 7.4, lOmM EDTA, lOmM NaCl,
100 ,ug/ml of Proteinase K). Mix well and vortex if
necessary. Add SDS (stock solution: 20%) to 1% final
concentration. Mix by gently inverting the tube. The sample
will turn very viscous. Place in rocker platform at 37 C
overnight with gentle mixing or at 60 C for 3 hours with
occasional mixing.
3o
-41
121S30~
1 6. Add NaC104 to 1 M final concentration from a
6 M stock (i.e. dilute 1:5). Mix gently by hand or in rocker
platform~ At this point, the sample can be stored in the
cold indefinitely.
7. Add equal volume of phenol-chloroform mix (1
part 90~ phenol, 10~ 1 M Tris pH 8.0 : 1 part CHC13) and
gently shake (e.g. wrist shaker) for 15 to 30 minutes at room
temperature.
8. Transfer to 15 ml glass Corex tube and
lO centrifuge at 4,000 rpm for 15 min. (beckman) or 10,000 rpm
for 5 min. (Sorvall) to separate the phases.
~. Remove ~o~ aqueous phase with wide mouth pipete
and return to original plastic tube. Repeat this extraction
procedure 2 more times.
10. Place DNA sample into an appropriately marked
dialysis bag and dialyze against 100 fold excess of TE buffer
(10 mM Tris pH 7.4, lmM.
11. Read O.D. of an appropriate sample dilution
(e.g. 1/20) against same type of blank solution at:
240nm (for EDTA); 250 nm (max. for DNA), 270 nm
(max for phenol); 280 nm (max. for proteins); 340 nm
~turbidity). 260/270 : approx. 1.2; 260/280 : approx. 1.8.
B. RESTRICTION ENDONUCLEASE DIGESTION OF GENOMIC
DNA:
1. Add the following components to a 1.5 ml
eppendorf tube:
a) Take appro,ximately 10,ug of DNA/test
(usually between 10 ~ul and 50 ,ul).
b) The appropriate amount of the specific 10X
3o endonuclease digestion buffer made to the
manufacturer's recommendations.
c) Restriction endonuclease in 3 fold excess.
-~2-
~21S3()4
l 2. Vortex 1 - 2 seconds or flick tube with finger
several times to mix.
3. Spin in eppendorf microcentrifuge l0 - 15
seconds to pellet reactants.
4. Incubate 2 hours at 37 C for Eco Rl or 65 C
for ~
5. a) Add 1/10 volume of 3M NH4 Acetate.
b) 2 to 2 l/2 ~olumes of cold 95~ EtOH.
c) Store at -20 C overnight. Spin in
microfuge ~o pellet 115 minutes at 4 C).
6. a) Dissolve pellet in 15 of H2O.
b3 Add the appropriate l~ X o~ restriction
enzyme buffer and a 3 fold excess of
restriction endonuclease and repeat steps
2, 3, and 4.
7. To stop reaction which is to be loaded to gel
immediately after digestion, add 5X ficoll marker dye
solution to a final concentration of lX. This can be done
with samples where the final volume is less than 20 ~l.
C. BLECTROPHORESIS
l. Prepare agarose gel by boiling agarose in lX
TAN (40mM Tris, pH 7.9; 4 mM NaAcetate, lmM EDTA). Final
concentration of agarose should be between 0.4~ and l.2%
depending on the size of the fragment to be fractionated.
Samples to be hybridized to pAW-l0l are electrophoresed in
0.4% agarose, while for hybridization to pLM 0.8 use l.2%
agarose.
2. When agarose solution reaches about 75 C, add
EtBr (2,7-diamino-l0-ethyl-9-phenyl-phenanthridinium bromide)
3o to a final concentration from 500 ng/ml to 12.5 ng/ml.
~21~
l 3. Immediately pour into a horizontal gel
electrophoresis mold to produce a gel approximately 4mm
thick. Place a well former at one end of mold. Allow to
cool at room temperature until solid. Remove well former and
5 cover ge~ with lX TAN.
D. Layer the samples into the gel wells. Connect
the gel box to the power supply. Turn on thP power supply
and dial up the current to the appropriate value. For
example, to separate fragments of over 10 kb, electrophorese
lO at 20 V for 3 days. For 1.5 kb fragments, electrophorese at
40 ~ overnight (16-20 hours) and after electrop~oresis,
disconnect the tank. Wearing gloves, remove gel with gel
scoop. Place gel on u.v. light box and lay a clear ruler
along side the lane with marker DNA. Take a picture of the
15 gel with an appropriate photographic film to keep as a record
of the electrophoresis,
D. PLASMID QUICK PREP
1. 15 ml of E. coli HB101 carrying either pAW101
or pLMO.8.
2. Centrifuge 10 min. at 8,000 rpm.
3. Pellet vortexed.
4 . Add 300 of 25% sucrose; 50mM Tris pH 8.0; 0.1
EDTA; 0.2 mg/ml RNase; 1 mg/ml Lysozyme.
5. Leave in ice for 15 min.
6. Add 250 0.5% Triton X-100; 50mM EDTA; 50mM
Tris pH 8Ø
7. Leave for 5 min. on ice.
8. Spin at 4 C for 30 min. 25K in SW-25, 27 or
41.
3o 9. Separate pellet from supernatant. (Pellet is a
gelation of bacterial DNA).
~Z~ 4
l 10. To the supernatant, add 10 of Proteinase K (5
mg/ml).
11. Leave 5 min. at R.T.
12. Extract once with l:l = phenol: CHCl3, twice
5 With C~IC13,
13. Aqueous phase add NH 4 Acetate to 0.3M final
concentrate.
14. Add 2.5 X vol. ethanol.
150 Leave in freezer (-20 C) overnight.
16. Centrifuge, dissolve the precipitate in 20mM
Tris pH 7.4, 10 mM EDTA.
17. Add CsCl for banding~
E. NICK TRANS~ATION
1. For each hybirdization reaction mix:
a) 50 nanograms of native probe DNA.
b) 0.7 ,ul of lOX nick translation buffer (lX =
25 mM Tris. HCl pH 7.9,
2.5 mM MgCl, 5 mM DTT, 100 ~g/ml of bovine
serum albumin).
c) 2.5 ul of alfa P-32 deoxynucleotides
triphosphate (25 yCi).
d) O. 5 ,ul of DNAse I at 20 picograms/,ul.
e) 0.5 ~l of DNA polymerase I (3 units).
Final volume is adjusted to 5 ~l.
2. Incubate at 16 C for 2 hours.
3. Stop reaction by adding ~DTA to a final
concentration of 10 mM and SDS to 0.5% final concentration.
Final volume 100 ~l.
4. Separate labeled DNA from unreacted
3o triphosphates by centrifugation of reaction mixture through
0.6 ml of SEpharose 6B-CL in a pierced microcentrifuge tube
at 1500 rpm for 2 min.
-45-
~53Q4
l 5. Take 1 ~1 of the flow through material (i.e.
containing the labeled DNA, and count in a beta scintillation
spectrometer.
F. SOUTHERN TRANSFER PROTOCOL FOR ZETAMIND
1. Run DNA on agarose gel. Stain with ethidium
bromide ~10 ~g/ml) for 15 to 30 min., remo~e excess stain by
soaking buffer for 15 to 30 min. and photograph.
2. Soak gel in 0.5 M NaOH, 1.0 M NaCl for 30 min.
with gentle agitation.
3. Rinse gel with water and repeat step 2 with 0.5
M Tris. HCl pH 7.5, 0.3 M NaC1,
4. Wet Zetabind with water. Then soak for 30 min.
in Na phosphate buffer ~0.025 M pH 6.5).
5. Soak gel for 15 min. in the same phosphate
15 buffer of step 4 for 20 min.
6. Place two strips of WHatman 3 MM wet in
phosphate buffer, the size of gel. Make sure that there are
no air bubbles trapped in between. Place gel (filters down)
over tray with a 3 MM paper wick submerged in phosphate
20 buffer. Place Zetabind on the gel, then two 3 MM paper
strips and finally paper towel (3-4 inches high). Place a
flat tray on top and some weight (e.g. a 100 ml bottle) to
ensure uniform contact between gel and papers.
7. Transfer overnight using phosphate buffer
(0.025 m pH 6.5).
8. Wash membrane with phosphate buffer for 15 min.
(rub gently the side of the membrane that was in contact with
gel).
9. Bake in vacuum oven for 2 hours at 80 C.
3o 10. Place in Seal-a-meal bag and wash for 30 to 60
min. at 60~ C in 0.1 X SCC, 0.5% SLS (approx. 15 ml).
-46-
~21S3Q4
l 11. Pour off buffer from step 10 and replace with
prehybridization buffer (4 X SSC, S0 mM Na phosphate pH 6.7,
5 X Denhardt, 200 ug/ml of denatured salmon sperm DNA and 50%
formamide). Incubate 3 to 16 hours at 37 C.
12. Denature the probe by heating in 1 ml of
hybridization buffer for 10 min. at 70 C. Hybridize with
the denatured radioactive DNA for 40 to 72 hours at 37
C~2xlO dpm/bag).
13. Wash with 2 X SSCP, 0.1~ SLS at 65 C agitating
lO for 20 min. until a 10 ml aliquot of the wash has less than
100 cpm Cherenkov (approx. 6 times). Wash twice with 0.4 X
SSCP, 0.02~ SLS at 65 C and twice with 0.1 X SSCP. Each
time add enough buffer to co~er filters.
14. Blot Zetabina and let air dry ~efore covering
15 with cellophane and placing in the cassette for
autoradiography.
Before reusing, remove probe by heating at 70 C
for 10 min. in prehybridization buffer.
20 PREHYBRIDIZATION (for 15 ml total volume)
-
1.5 ml denatured salmon sperm DNA (5 mg/ml)
3.0 ml 20 X SCC
1.5 ml 50 X Denhardt
1.5 ml 0.5 M phosphate
7.5 ml 100% formamide
0.15 ml 20% SLS
.
3o
32p _ labeled denatured DNA
1.5 ml more of 20 X SSC
\ ~
:~2:1~;304
1 HYBRIDIZAITON (for 15 ml total volume).
15. The 6 strips from two gels are taped on yellow
paper from an x-ray film pack, labeled, covered with plastic
` wrap and placed in cassette with built in intensifying
screens. In dark room, the cassette is loaded with 8 X 10
inch X-omat AR x-ray film placing fllm between nitrocellulose
strips and screen. ~he cassette is closed and placed in a
freezer at -70 C.
16. The x-ray is de~eloped in 24 to 48 hours. The
film is removed from cassette and deeloped in dark room with
yellow safe light on. The cassette may be reloaded if
another exposure is required.
3o
--48--
~z~sa~
EXAMPLE VI
This Example illustrates the specific performance
and evaluation of a paternity test employing the subject
invention.
1. Blood samples are taken from the mother, child
and putative father and DNA purified as described in Example
V A.
2. These DNAs are separately reacted with either
restriction enzyme EcoRl or Taq 1 as described in Example V
10 B-
3. These DNAs are subjected to electrophoresis as
described in Fxample V C using 5 ~g of one of the three DNAs
in each of three adjacent lanes in order (from left to right)
mother, child putative father.
4. "Paternity Probe" DNA' s are prepared and
labelled as described in Examples V D and V E.
5. The electrophoreses DNAs are blotted as
described in Example V F.
6. The labelled probe DNAs from step 4 are
20 hybridized with the blotted genomic DNAs from step 5 as
described in Example V E. pAW 101 DNA is hybridized to EcoRl
cut genomic DNA while pLM 0.8 is hy~ridized to ~ 1 cut
genomic DNA .
7. Autoradiograms are made as described in Example
25 V F.
8. Following autoradiography, the size of the
bands corresponding to the polymorphic DNA fragments are
determined, This accomplished by measuring the distance
migrated by these bands, relative to that of a collection of
3o D~A molecular weight standards (Southern, E.M., 11984] Anal.
Biochem. 100, 319-323).
The size of the DNA fragments, in each of the
-49-
~S3~4
1 individuals of a family, are compared and used to determine
whether the pattern observed in the child is consistent with
those measured in the putative father. If the size of the
DNA fragments i the child are different to that of the
5 presumptive father, then it is concluded that he is not the
biological father (i.e. case of non-paternity~. If the child
shares only one allele with the mother then it can be
concluded that the other allele was inherited form the
father. If the putative father does not possess this allele
10 it can be concluded that he is not the father.
Alternatively, if the two share at least a pair of DNA
fragments, not co~tri~uted by the mother than the
determination ~f whether or not that individual might be the
father is based on the probability that a random indivi~al
from the population might have that same DNA fragment size
(i.e. paternity index; in Inclusion Probabilities in
Parentage Testing [1983] , ed. R. H. Walker, American
Association of Blood Banks). In this latter case it is
necessary to know the frequency of the alleles detected with
the particular DNA probe. The observed frequencies for the
probes pAW-101 and pLM-0.8 are given in tables 1 and 2.
3o
--50--
~21~i~4
EXAMPLE VI I
Test Case 1
Using the procedures of Example VI a mother, child
and putative father were tested using the subject invention.
5 Figure 1 shows a picture of the autoradiogram obtained using
pAW101 as a probe against EcoR1 cut DNA obtained format he
mother, child and father. Measurement of migration distances
and comparison with known standards indicated that the mother
carries pAW101-alleles number 2 and 5, the child carries
10 pAW101-alleles number 5 and 10 while the putative father
carries pAW101-alleles numbers 10 and 11. Since the mother
must have contributed pAW101-allele number 5 to the child the
father must have contributed allele number 10. One now can
compare the chance that the putative father would contribute
15 pAW101 allele number 10 to a child vs the chance that a
random man would contribute allele number 10. In this case,
the likelihood ratio if 16.67 which translates into a chance
of paternity of 94%.
3o
-51-
1215;~04
1 EXAMPLE VIII
Test Case 2
Using the procedures of Example VI, a mother, child
and putative father were tested using the subject in~ention.
Fiqure 2 shows a picture of the autoradiogram obtained using
pAW101 as a probe against EcoRl cut DNA obtained from the
mother~ child and father. Measurement of migration distance
and comparison with known standards indicated that the mother
carries pAW101-alleles number 5 and 9, the child carries
10 pAW101-alleles numbers 5 and 7 while the putative father
carries pAW101-alleles number 4 and 6. Since the father of
this child must have contributed allele 7 to the child and
the putative father does not carry this allele, he is
excluded as a possible father.
3o
S;~)4
1 EXAMPLE IX
Test Case 3
Using the procedures of Example VI, a mother, child
and putative father were tested using the subject invention.
5 Figure 3 shows a picture of the aut~radiogram obtained using
pLM ~.8 as a probe against Taq 1 cut DNA obtained from the
mother, child and father. Measurement of migration distances
and comparison with known standards indicated that the mother
carries pLM 0.8-alleles number 7 and 8, the child carries pLM
10 ~.8-alleles number 7 and 8 while the putative father carries
pLM 0.8-alleles numbers 2 and 8. Since the mother could have
contributed either pLM 0.8 allele number 7 or 8 to the child
one can only conclude that the father must have contributed
either allele 7 or 8. One can compare the chance that
15 putative father would contribute either pLM 0.8 allele 7 or 8
to a child vs the chance that a random man would contribute
either of these alleles. In this case the likelihood ratio
is 3.55 which corresponds to a chance of paternity of 71.8%.
3o
-53-
~53Q4
Table I
Fre~uency of alleles visualized using pAW101 as a
probe and Eco~l cut human genomic DNA in a population of 298
- random individuals.
Allele # Size Frequency
(in kilobase pair)
1 14.0 0.013
2 14.5 0.052
3 14.9 0.077
4 15.4 0.117
16.0 0.1~6
6 16.6 0.117
7 17.2 0.064
8 17.7 0.040
9 18.3 0.035
lg.0 0.030
11 19.6 0.035
12 20.2 0.040
13 20.8 0.064
14 21.6 0.069
22.2 0.023
16 22.7 0.018
17 23.6 0.020
18 24.3 0.003
19 24.6 0.008
25.3 0.013
21 26.1 0.008
22 27.1 0.002
3o 23 28.1 0.002
-54-
~;~15304
Table 2
Frequency of alleles visualized using pLM 0.8 as a
probe and EcoRl cut human genomic DNA in a population of 268
- random individuals.
Allele # Size Frequency
(in kllobase pair)
1 2.35 0.089
2 2.65 0.580
3 2.75 0.041
4 2.95 0.009
3.08 ~.123
6 3.40 0.0~7
7 3.70 0.123
8 4.09 0.018
9 4.30 0.007
3o
