Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2013430
NETHOD F9R IDENTIFYING H~MAN BUBJECTS BY
ANALYBIS OF GENETIC MATERIAL ~ -
Field of the Invention
This invention pertains to the use of specific
5 oligonucleotides as probes for determining the source or -
identity of human biological material. -
Background of the Invention
The human genome contains approximately 50~000
copies of an interspersed nucleotide repeat with the
seq~ence (dT-dG:dA-dC)n, where n is an integer from about
10 to 60. Several of these repeats have variable lengths ~ ;
in different individuals, where allelic fragments of the
repeats vary in size by multiples of 2 base pairs (bp) or
more. Litt and Luty, Am. J. Hum. Genet. 44:397-401 -
(1989); Weber and May, ~m. J. Hum. Genet. (Sup~l.) 43:A161 ~`
(1988). Generally, such repeats display extensive length ~
polymorphism in humans as variable numbers of tandem `
repeats (VNTRs).
Many highly polymorphic VNTRs found to date in
human genomes tend to be distributed mainly in
subtelomeric regions of human chromosomes. Unfortunately,
genetic information located in such regions can be
difficult to retrieve without performing haplotyping or ;
other difficult procedure and difficult to analyze
accurately.
Summary of the Invention
In an attempt to circumvent this problem, a
search was made for highly polymorphic (TG) n .,
microsatellite regions in interstltial regions of
chromosomes.
A number of clones previously mapped to
interstitial chromosomal regions were searched to
ascertain if the clones also included highly polymorphic
loci. A locus was found, designated DllS35, that
contained at least one polymorphic (dT-dG) n microsatellite
region. The DllS35 locus had been previously mapped to
region llq22 on human chromosome 11. Maslen et al.,
Genomics 2:66-75 (1988). The DllS35 locus was found to
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have at least 6 alleles and a heterozygosity of nearly
90~. This finding suggested that highly polymorphic (TG) n
microsatellites may be readily ascertained in interstitial
genomic regions.
5Because the full information content of
interstitial polymorphic loci such as DllS35 is more
accessible for genetic analysis, these loci are especially
useful, for example, in mapping genes for late-onset
diseases and syndromes via affected sib-pair and affected
relative-pair analyses.
To resolve the VNTRs at the DllS35 locus, a
sample of human DNA i~ first denatured to form single- -
ctranded DNA. After denaturation, the human DNA is ~ -~
annealed with two unique single-stranded oligonucleotide
primers. The first primer is homologous with a portion of
a unique DNA sequence on a first DNA strand flanking a
first end of the (dT-dG)n microsatellite region at the
DllS35 locus. The second primer is homologous with a
portion of a unique DNA seguence on a second strand
flanking a second end of the same (dT-dG)n microsatellite
region. After annealing, the primers are extended using
the polymerase chain reaction (PCR) technique using ~; ~
radiolabeled DNA precursers. Plural PCR cycles including : ;
heat denaturation of the DNA, annealing of the primers to ;
25 their complementary sequences on the human DNA, and ;~
extension of the annealed primers with DNA polymerase are ; ~;
employed to enormously increase the numbers of copies of
the (dT-dG)n microsatellite repeat sequences adjacent the
hybridized primers. The amplified copies are then
electrophoresed on sequencing gels for sizing and
interpretation of the resulting pattern. ~;
The preferred oligonucleotide primers, designated -~
oligomers 780 and 781, were custom-synthesized as
described herein. Each primer had a known sequence 20 ;-; -
nucleotides long, where each sequence corresponded to a
portion of the sequence of a DllS35 locus present in the ~
genome of a recombinant phage. Although oligomers 780 and ;
781 are the preferred primers, other single-stranded DNA
20~3~30
oligomers having nucleotide sequences homologous to other
portions of the unique sequences flanking the (dT-dG)n
microsatellite repeat region at the DllS35 locus can al~o
be used, where each primer has a length within the range
of about 15 to about 30 nucleotides long.
Hence, one feature of the present invention
comprises a method for detecting microsatellite ~ -~
polymorphisms in DNA from a sample of human individuals
selected from a population of individuals. Steps in the
method include isolating DNA from each individual in the
sample; heat-denaturing the DNA; annealing single-stranded
DNA oligomeric primers to the denatured DNA, wh~re the
oligomeric primers are homologous to unique nucleotide
sequences flanking the microsatellite (dT-dG) n repeats at
the DllS35 locus; performing multiple cycles of the
polymerase chain reaction on the primed DNA to amplify
copies of microsatellite DNA sequences located adjacent
the hybridized primers; and separating the amplified
copies of DNA for visualization and interpretation. The
amplified seguences comprise (dT-dG) n repeat sequences at
the DllS35 locus on human chromosome 11. This method can
be used, for example, for ascertaining a person's pedigree
or genetic identity.
As another feature of the present invention, the
method as described hereinabove can also be used for
determining the identity of a human subject, wherein the
human su~ject is suspected of being the source of a sample
of biological material of unknown origin. Such a method
has especial utility in forensic investigations.
As another feature of the present invention, the
step of using the polymerase chain reaction instead of
using Southern Blotting or related techniques renders the
present much faster to perform than existing techniques of
genetlc typing. Further, use of DNA sequencing gels to
1 35 resolve amplified sequences facilitates the speed of the
i present method over existing methods.
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As another feature of the present invention,
primers 780 and 781 have been sequenced as a preliminary
step to custom-synthesis of each primer.
Brief Description of the Drawina ~-
FIG. 1 shows the nucleotide sequence of a 196 bp
region of the phage ~2-22 subclone including a ~T-G) 17
repeat used to design oligonucleotide primers used in the
present invention.
FIG. 2 is an autoradiogram of a sequencing gel
used to resolve PCR-amplified segments from genomic DNAs
from members of a three-generation family with autosomal ;
dominant episodic ataxia, showing the allelic
characteristic of the microsatellite (dT-dG) n repeat at
the DllS35 locus, as determined via the present method.
Detailed Descrie~ion ~ ;~
The preferred flanking primers used herein were ;;
designated oligomers 780 and 781. Each was a synthetic
DNA oligomer 20 nucleotides (nt) long. These oligomers
were designed to have sequences complementary to portions
of unigue genomic sequences flanking a (dT-dG) n
microsatellite region at the DllS35 locus. The DllS35
locus is normally located on human chromosome 11.
After hybridization to a sample of human DNA
containing the DllS35 locus, oligomers 780 and 781 were
used as primers for multiple cycles of the polymerase
chain reaction (PCR). The PCR produced a selective
amplification of the (dG-dC)n-containing sequences of at
least a million fold. The PCR technique is described
generally in Saiki et al., Science 239:487-491 (1988).
' 30 Oligomers 780 and 781 were synthesized as
follows: ~-
Using as a hybridization probe a commercially
available alternating copolymer of poly-(dG-dT):poly-(dC-
dA), a number of recombinant phage clones containing DNA
from the long arm of human chromosome 11 were screened.
See Frischauf et al., J. Mol. Biol. 170:827-842 (1983) for
details on constructing the llq;16q chromosome-specific -~
library using the lambda replacement vector EMBL-3. The ~` ;
.! ~
2~34~
screened recombinant phages used as described herein
originated from such a library. See also Maslen et al.,
Genomics 2:66-75 (1988). ~ -
The phage ~2-22, previously mapped to the DllS35
locus on the llq22 chromosomal region, Maslen et al.,
Genomics 2:66-75 (1988), gave a strong signal when
hybridized with the poly (dG-dT):poly (dC-dA) probe. To
perform these analyses, slot blots were performed wherein
each slot contained 10 ng DNA from several selected clones
of chromosome llq lambda phage. Maslen et al., Genomics
~:66-75 (1988). Each slot was probed with nick-translated
poly (dG-dT):poly (dA-dC) (Pharmacia). Hybridization was
performed overnight at 65C in 0.5 M sodium phosphate, pH
7.0, 7 % (w/v) SDS, 1 % (wlv) bovine serum albumin, where
the solution lacked nonradioactive DNA. Church and
Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995 (1984).
Filters were washed at a maximum stringency of 0.1 X
SSC/O.l % SDS at 55C. Overnight autoradiography
indicated that phage ~2-22 (DllS35) gave a strong signal.
A Sau 3A digest of phage ~2-22 was subcloned into
the Sal 1 site of the plasmid vector pTZ18u. Colony
filters containing subclones were probed with nick-
translated poly (~G-dT):poly (dA-dC) as described above to
identify subclones containing (TG) n microsatellites. One
subclone found to contain (TG) n microsatellites was
sequenced using a standard dideoxynucleotide sequencing
protocol. Sanger et al., Proc. Natl. Acad. Sci. USA
74:5463-5467 (1977). The sequence of a 196 bp region of
the ~2-22 subclone inc~uding a (T-G) 17 repeat (underlined)
is shown in FIG. 1. Twenty-nucleotide segments flanking
this repeat, shown boxed, were used to design oligomers
780 and 781. In choosing these specific segmen~s, the
following criteria were considered in descending order of
importance: (1) sequences containing four or more
contiguous purines or pyrimidines or other simple
sequences should be absent; (2) the GC contents of the two
primers should match within +/- 2 nt; and (3) the primers
should flank the (TG)n repeats as closely as possible.
20~3~
-- 6
Although primers 780 and 781 were deemed to be
the preferred primers derivable from the sequence shown in
FIG. 1, primers having other unique sequences flanking the
(dT-dG) n microsatellite repeat at the DllS35 locus are
also possible. Generally, primers as used in ~he present
invention should have a single-stranded length within the
range of about 15 nucleotides to about 30 nucleotides, and
preferably about 20 nucleotides long. Shorter primers
tend to exhibit lesser specificity when hybridizing to
human DNA; longer primers require higher hybridization
temperatures and longer hybridization times. Also, longer
primers are more expensive to custom-synthesize.
Using the sequence as shown in FIG. 1 as a guide,
20-nt primers 780 and 781 flanking the (TG)n
microsatellite repeat region were synthesized on an
Applied Biosystems Model 380A DNA Synthesizer and purified
on a 12% denaturing acrylamide gel. The oligomer primer
sequences were:
Oligomer 780: 5' ACA ATT GGA TTA CTA CTA GC 3'
Oligomer 781: 5' TGT ATT TGT ATC GAT TAA CC 3'
These primers were used to amplify the (dT-dG) n
microsatellite region of locus DllS35 in samples of
genomic DNA from family members and unrelated individuals.
Genomic DNA is prepared as described by Litt and
White, Proc. Natl. Acad. Sci. USA 82:6206-6210 (1985).
After denaturation of the DNA to form single-stranded DNA, ~-
the PCR method of Saiki et al. is used to amplify the (dT-
dG) n microsatellite xepeat regions at DllS35 loci in the
DNA. Amplifications were performed using 250-ng samples
of genomic DNA. To perform the PCR, the thermostable Taq
1 DNA polymerase and additional reagents supplied in the
GeneAmpT~ kit (Perkin Elmer-Cetus, Norwalk, CT) were used.
~ 35 Reaction mixtures were prepared according to the
I instructions of the manufacturer, except that the total
volume was decreased to 25 ~L and 3-5 ~Ci of ~-[32P]dCTP
was included in each reaction tube.
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The PCR amplification method comprises multiple
cycles of heat-denaturing the DNA, annealing the primers
to their complementary sequences on the denatured DNA, and
extension of the annealed primers using a DNA polymerase.
The preferred DNA polymerase is the thermostable DNA
polymerase purified from the thermophilic ~acterium
Thermus aquaticus (~) which is stable to about 95C.
The required number of amplification cycles can
vary, depending upon the amount of genomic DNA in the
original sample and the amount of DNA required for
subsequent analysis of the length distribution of the
amplified portions thereof. In one test, thirt~v-seven
cycles of amplification were performed manually wherein
each cycle included a denaturation step for 1 min at 90-
92C, an annealing step for 2 min at 40C, and a primer
extension step for 2 min at 72C. The thirty-seven
amplification cycles produced a sufficient amount of
amplified sequences for visualization thereof on a DNA
sequencing gel.
To separate the amplified sequences for
subsequent length-distribution analysis, aliquots of the
DNA were electrophoresed on a DNA sequencing gel in
alternate lanes. After electrophoresis, gels were
autoradiographed for 1-3 days, usually without
intensifying screens. Segment lengths were measured
relative to size standards consisting of end-labeled Sau
3A fragments of pBR322 and/or DNA sequence ladders derived
from a known sequence.
Under conditions as described hereinabove,
oligomers 780 and 781 amplified polymorphic microsatellite
(dT-dG) n sequences at DllS35 loci having at least six
different alleles as seen in 17 unrelated individuals. As
shown in Table 1, fifteen of these 17 individuals (88%)
were heterozygous. Using the allele frequencies shown in
Table 1, a polymorphism information content (PIC) value of
0.79 was calculated. Botstein et al., Amer. J. Hum.
- Genet. 32:314-321 (1980).
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TABLE 1
Allele Size (nt) Number
1 162 4
2 160 9
5 3 158 5
4 156
154 6
6 152 6
FIG. 2 shows an autoradiogram of a sequencing gel
used to resolve (dT-dG)n microsatellite repeats amplified ~ ;
from yenomic DNAs from members of a three-generation
family. The family has a history of autosomal dominant
episodic ataxia. Each lane contains one or two relatively
intense "major'l bands where each major band is closely
associated with a cluster of up to eight less-intense
minor bands spaced at one-nucleotide intervals.
Considering that each of the major bands represents an
allele, codominant Mendelian inheritance is observed in
the family shown in FIG. 2. Another study of three
nuclear families with a total of 22 children confirmed
that the polymorphism was inherited in a Mendelian fashion
(data not shown).
Within a set of unrelated individuals, the value
of n in the (dT-dG) n microsatellite repeat at the DllS35
locus was found to vary extensively. Indeed, as disclosed
above, oligomers 780 and 781 revealed a highly polymorphic
locus in human DNA with a heterozygosity of 88%. Hence,
this pair of oligomers has utility in paternity testing
and in other forensic procedures, such as identifying or
ruling out individuals suspected of rape, tracing the
source of a bloodstain, and identifying a badly disfigured
corpseO In particular, any sample of biological material
containing human DNA can be tested using the present
method and oligomers 780 and 781 to help determine the
origin of the sample. The present method is particularly
useful with small samples because the PCR step amplifies
the polymorphic sequences of interest to a concentration
2~3~3~
g
sufficient for conventional analysis using gel
electrophoresis.
The preferred method for detecting (TG)n
microsatellite polymorphisms is via th~ use of primers 780
and 781 and the polymerase chain reaction (PCR) to amplify
the repeat-containing region of interest located between
the primer-annealing sites in genomic DNA. This has major
advantages over the conventional Southern blotting
technique used to detect restriction fragment length
polvmorphisms (RFLPs) that are often used for forensic
purposes. In particular, the new method as described
herein is far more sensitive than Southern Blotting. In
particular, 250-ng samples of human DNA have been used in
the present method which is tenfold lower than required
for Southern blotting. Since others have used the PCR
method to amplify extremely small quantities of human
genomic DNA present in a single cell, it is very likely
that, in the present method, the required sample size for
analysis could be decreased hy at least several orders of
magnitude, if necessary. Another advantage is that the
technology described seems to be faster and more reliable
than Southern Blotting.
As shown in Table 1, only six alleles have been
found to date at the DllS35 locus. As a result, there is
a small but calculable probability that two unrelated
individuals having genomes probed using primers 780 and
781 will display the same genotype. This limitation can
be minimized by using additional primer pairs which detect
other highly polymorphic (TG) n microsatellites, such as
primers 635 and 636. Litt and Luty, Am. J. Hum. Genet.
44:397-401 (1989). Since there is no overlap between
segment sizes observed with these two primer pairs, it
should be feasible to type individuals for both
polymorphic loci by co-amplification of genomic DNAs with
the products resolved on a single gel.
The results described hereinabove indicate that
the DllS35 locus, previously mapped to llq2~l contains a
microsatellite VNTR with at least six alleles and a PIC of
;
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about 0.78. These results also indicate that highly
polymorphic (TG) n microsatellite VNTRs can be readily
ascertained in genomic DNA regions where "classical"
VNTR's are scarce. Interestingly, another research group
has described microsatellite VNTRs at interstitial genomic
regions on other human chromosomes: APOA2 (PIC = 0.65;
lq21-q23) and APOC2 (PIC = 0.79; l9ql2-ql3.2). Weber and
May, Amer. J. Hum. Genet. Sup~l. 43:A161 (1988). Because
their full information content is inaccessible without
haplotyping, VNTRs in interstitial chromosomal regions are
especially useful in genetic mapping of "late onset"
disorders, such as Alzheimer's disease, via affected sib
pair and affected relative pair analyses.
Having illustrated and described the principles
of the present invention, it should be apparent to those
of ordinary skill in the art that such embodiments may be
modified in detail without departing from such principles.
I claim as my invention all such modifications as come
within the true spirit and scope of the following claims.
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