U.S. patent application number 09/840717 was filed with the patent office on 2002-10-24 for method for genotyping microsatellite dna markers.
Invention is credited to Belouchi, Abdelmajid, Paquin, Bruno.
Application Number | 20020155449 09/840717 |
Document ID | / |
Family ID | 25283033 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155449 |
Kind Code |
A1 |
Belouchi, Abdelmajid ; et
al. |
October 24, 2002 |
METHOD FOR GENOTYPING MICROSATELLITE DNA MARKERS
Abstract
The present invention provides a high throughput method for
genotyping microsatellite markers. The technology uses a
combination of oligonucleotides in a selective ligation reaction
that discriminates mono-, di-, tri-, tetra-, penta-, hexa-, hepta-,
octa- and nona-nucleotide repeated alleles in amplified DNA from
individuals.
Inventors: |
Belouchi, Abdelmajid;
(Montreal, CA) ; Paquin, Bruno; (Chateauguay,
CA) |
Correspondence
Address: |
GIBBONS, DEL DEO, DOLAN, GRIFFINGER & VECCHIONE
1 RIVERFRONT PLAZA
NEWARK
NJ
07102-5497
US
|
Family ID: |
25283033 |
Appl. No.: |
09/840717 |
Filed: |
April 23, 2001 |
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6858 20130101; C12Q 2535/125 20130101; C12Q 2535/107
20130101; C12Q 2537/143 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for genotyping different of alleles of a microsatellite
DNA locus by using combinations of at least three oligonucleotides
for each allele on the locus comprising: (a) providing a sample
containing the microsatellite DNA; (b) selecting at least three
oligonucleotides comprising: (i) a 5' primer which comprises at
least a 5 base pair sequence that is complementary a flanking
region of a repeat region of the microsatellite; (ii) a central
primer which is complementary to a repeated region of the
microsatellite DNA; and; (iii) a plurality of 3' primers which
comprises: (a) a sequence that is complementary to the 5' flanking
sequence of the repeated region of the microsatellite; and (b) a
number (n) of repeat units at the 5' end of the plurality of 3'
primers; (c) mixing the sample and primers such that the primers
and microsatellite DNA hybridize; (d) adding a ligating reagent;
and (e) detecting the presence of ligation products that consist of
all the oligonucleotide primers.
2. A method for genotyping different of alleles of a microsatellite
DNA locus by using combinations of at least three oligonucleotides
for each allele on the locus comprising: (a) providing a sample
containing the microsatellite DNA; (b) selecting at least three
oligonucleotides comprising: (i) a 5' primer which comprises of a 5
base pair sequence that is complementary a flanking region of a
repeat region of the microsatellite; (ii) a central primer which is
complementary to a repeated region of the microsatellite DNA; and;
(iii) a plurality of 3' primers which comprises: (a) a sequence
that is complementary to the 5' flanking sequence of the repeated
region of the microsatellite; and (b) a number (n) of repeat units
at the 5' end of the plurality of 3' primers; (c) mixing the sample
and primers such that the primers and microsatellite DNA hybridize;
(d) adding a ligating reagent; and (e) detecting the presence of
ligation products that consist of all the oligonucleotide
primers.
3. The method according to claim 2, wherein the sample is a mixture
of different samples containing different alleles of a given
microsatellite DNA
4. The method according to claim 2, wherein the microsatellite DNA
comprises of mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-
and nona-nucleotide repeated alleles.
5. The method according to claim 2, wherein the sample is an
amplified PCR fragment of the microsatellite DNA.
6. The method according to claim 2, wherein the oligonucleotides
comprise of modified nucleosides.
7. The method according to claim 2, wherein the selecting step
further comprises labeling the 5' end of the 5' primer with a
detectable label.
8. The method according to claim 2, wherein the detecting step
comprises separating the ligation products according to their size
by gel electrophoresis.
9. The method according to claim 2, wherein the ligation reagent is
T4 DNA ligase or a thermostable ligase.
10. The method according to claim 2, wherein the detecting step
further comprises separating the ligation products by a gel free
system wherein the gel free system comprises covalently linking a
functional group to the 5' end of the 5' primer wherein the
functional group exhibits specific binding to a component of a
solid support; and labeling the 3' end of the 3' primers with
different detectable labels wherein the different labels yield
different signals.
11. The method according to claim 10, wherein the detectable labels
are fluorescent labels.
12. The method according to claim 10, wherein the functional group
is biotin and the solid support is coated with streptavidin.
13. A kit for detection of the presence of different microsatellite
DNA on a loci comprising combinations of at least three
oligonucleotides and instructions for use of the oligonucleotides
in a suitable container means, wherein the oligonucleotides
comprise sequences which are complementary to and hybridize with
one strand of the repeat region of a microsatellite DNA and having
terminal groups such that the oligonucleotides are ligatable to
each other when hybridized to the microsatellite DNA.
14. The kit according to claim 13, wherein at least of one of the
oligonucleotides is labeled with a detectable label.
15. The kit according to claim 13, wherein the label is a
fluorescent label.
16. The kit according to claim 13, comprising: (a) a 5' primer; (b)
a central primer; (c) a plurality of 3' primers; and (d) a ligating
reagent.
17. A kit for detection of the presence of different microsatellite
alleles at a locus by separating the ligation products by a gel
free system comprising combinations of at least three
oligonucleotides and instructions for use of the oligonucleotides
in a suitable container means, wherein the oligonucleotides
comprise at least one primer that is covalently-linked to a
functional group, wherein the functional group exhibits specific
binding to a component of a solid support and at least one other
primer is labeled with different detectable labels, wherein the
different labels yield different signals.
18. A kit according to claim 17, comprising: (a) a 5' primer
wherein the 5' is covalently-linked to a functional group at the 5'
end of the 5' primer; (b) a central primer; (c) a plurality of 3'
primers wherein the 3' end of the 3' primer is labeled; (d) a
ligating reagent; and (e) a solid support
19. The kit according to claim 17, wherein the different detectable
labels are different fluorescent labels.
20. The kit according to claim 17, wherein the functional group is
biotin and the solid support is coated with streptavidin.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for genotyping
microsatellite DNA markers using ligation of at least three
oligonucleotides. Specifically, the present invention provides a
method for distinguishing allele content in mono-, di-, tri-,
tetra-, penta-, hexa-, hepta-, octa- or nona- repeated DNA using
combinations of at least three oligonucleotides comprising a 5'
primer, a central primer and a plurality 3' primers that hybridize
to different alleles of microsatellite DNAs.
BACKGROUND OF THE INVENTION
[0002] The analysis of variation among polymorphic DNA provides
valuable tools for genetic studies in the development of genetic
engineering, medicine, gene mapping and drugs. For example,
variations in polymorphic DNA allows one to distinguish one
individual of a population from another, or to assess the
predisposition of an individual to a heritable disease or
trait.
[0003] Two types of genetic markers widely used in genetic studies
include microsatellites and single nucleotide polymorphisms (SNPs).
Microsatellites are genomic regions that are distributed
approximately every 30 kilobases throughout the genome and that
contain a variable number of tandemly repeated sequences of mono,
di-, tri-, tetra-, penta-, hexa-, hepta-, octa- or
nona-nucleotides. SNPs are found approximately every kilobase in
the genome.
[0004] SNPs and microsatellites differ in primary DNA structure,
relative genome density and genetic information. For example, SNPs
are more suitable than microsatellites for genotyping with a
high-density of markers because of their distribution and the high
sequence specificity possessed by sequences adjacent to the SNP
site. Yet, microsatellites are more informative than SNPs because
microsatellites typically possess four to sixteen different alleles
compared to only two alleles for SNPs.
[0005] Presently, the most commonly used methods for genotyping
microsatellite markers are gel-based PCR fragment analysis (Shi et
al., (1999) Mol. Diagn., 4: 343-351). These methods are relatively
more labor-intensive and time-consuming due to gel preparation and
gel reading steps, than the method described in this invention.
Moreover, the automated DNA genotyping instruments are expensive
compared to other forms of detection and do not address the gel
reading problems resulting from nucleotide compression. Other
methods such as differential hybridization are limited by
hybridization to two or more microsatellite markers that share
sequences. (see Korkko et al., 1998).
[0006] Oligonucleotide Ligation Assays (OLAs) have been used to
detect SNPs (Baron et al., 1996, see also U.S. Pat. Nos. 5,242,794
and 5,866,337) or mutations in a gene (Landegren et al., 1988, U.S.
Pat. Nos. 4,988,617 and 6,025,139). These OLAs are designed to
hybridize contiguously to single-stranded target DNA sequences.
Recently, an OLA was developed to genotype microsatellites
containing mono- and dinucleotide repeats (Zirvi et al., 1999a,
1999b, U.S. Pat. No. 6,054,564, WO 98/03673, EP956359).
[0007] FIG. 1 demonstrates the methods employed in these previous
OLA methods. For example, in the OLA method used for the detection
of SNPs, two oligonucleotides are designed to hybridize to the
region of the tested site where ligation would occur. The principle
in the OLA assay for detection of mutations within a gene is to
hybridize multiple short oligonucleotides contiguously throughout
the entire gene. An OLA method for genotyping mono- and
dinucleotide repeats has been reported where the ligation of the
two oligonucleotides was performed at the middle of the repeat. In
all of these methods, the presence of a mismatch would prevent
hybridization and ligation of the oligonucleotide at or near the
location of the mismatch.
[0008] The major drawback of using OLA for genotyping
microsatellites is that ligation is not a highly discriminating
process and background noise can be a significant problem. To
circumvent this problem, modified nucleotides (containing
nucleoside analogs) near the ligation junction are used to improve
the stringency of both the hybridization and the ligation. However,
this raises the cost, because relatively long, specific
oligonucleotides are required for these assays. A method that
overcomes these disadvantages of OLA would make this approach
simpler and more efficient and amenable to the comparative
genotyping of pooled DNA samples.
SUMMARY OF THE INVENTION
[0009] The present invention relates to a method for genotyping
different microsatellite DNA markers using ligation of at least
three oligonucleotides. In previous inventions, the principle was
to hybridize combinations of two, long oligonucleotides
contiguously in order to cover the whole sequence of the
microsatellite. This requires the costly synthesis of specific,
long oligonucleotides for each microsatellite to be genotyped. In
addition, the use of modified nucleotides was necessary to achieve
specificity. The present invention has eliminated these problems by
using combinations of at least three oligonucleotides for each
allele at a locus. Collections of limited numbers of
oligonucleotides can be used for genotyping many different
microsatellites, thus reducing the cost of oligonucleotide
synthesis in large genotyping projects. In addition, by using
combinations of three oligonucleotides, a high degree of
specificity is achieved without the need to use modified nucleoside
analogs.
[0010] The present invention comprises the steps of providing a
sample containing microsatellite DNA; selecting combinations of at
least three oligonucleotides that comprise a 5' primer, a central
primer and a 3' primers; mixing the sample and primers such that
the primers and microsatellite DNA hybridize; adding a ligating
reagent; and detecting the presence of ligation products that
consist of combinations of three oligonucleotides (5' primer,
central primer and 3' primers) bound together as a single
oligonucleotide with a contiguous sequence, reflecting the precise
genotype of the microsatellite in the sample, and thus the
allele(s) present. In particular, the 5' primer comprises at least
5 base pairs complementary to the 3' flanking region of the
microsatellite target strand; the central primer is complementary
to the repeated region of the microsatellite target strand DNA; and
the 3' primers comprise sequences that are complementary to the 5'
flanking sequence of the microsatellite target strand with the
addition, at the 5' end, varying numbers of repeat units. The
nature and the number of repeat units comprising the central primer
depend on the nucleotides in the repeated sequence and the number
of repeat units of the shortest allele at a given locus. Likewise,
the number of repeat units added at the 5' end of the 3' primer
also depends on the nucleotides in the repeated sequence and the
number and identity (length) of the alleles in a population.
[0011] The present invention further provides a method for
genotyping a DNA pool containing a mixture of different DNA samples
of the same microsatellite wherein the microsatellite DNA includes
mono-, di-, tri-, tetra- penta-, hexa-, hepta-, octa- or
nona-nucleotide repeated alleles. The detection of the allele
content of the microsatellite DNA marker within the pooled sample
is determined by gel filtration, electrophoresis, mass spectrometry
or a gel free analysis.
[0012] In one embodiment, the present invention provides a method
for genotyping different alleles of a microsatellite DNA wherein
the 5' end of the 5' primer is labeled with a detectable label. In
another embodiment, the method provides detection of different
allele of a microsatellite DNA wherein the 5' or 3' primer is
covalently linked to a functional group, wherein the functional
group is capable of specifically binding to a component of a solid
support.
[0013] The present invention will decrease cost and improve the
experimental quality needed to achieve genotyping using high
density DNA pooling. The method uses the capacity of DNA ligase to
join selectively designed adjacent oligonucleotides that hybridize
to a given DNA template. The combination of specifically designed
oligonucleotides for each allele within a marker will allow
discrimination between the different genotypes.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1: Prior Art Oligonucleotide Ligation Assay (OLA)
Techniques
[0015] FIG. 1 shows other prior techniques for OLA. In particular,
the top figure demonstrates an assay for the detection of mutations
within a gene. (Landergren et al. 1988; U.S. Pat. Nos. 4,988,617
and 6,025,319). The middle figure demonstrates an OLA for the
detection of SNPs. (Baron et al.; U.S. Pat. Nos. 5,242794 and
5,866,337). The bottom figure demonstrates an OLA for genotyping
mono- and dinucleotide repeats. (Zirvi et al., 1999a, 1999b, U.S.
Pat. No.: 6,054,564). The template is a single-stranded DNA
containing the complementary sequence which the oligonucleotides
are hybridizing. The letter "L" points the ligation sites.
[0016] FIG. 2: Oligonucleotide Ligation Assay Principle
[0017] FIG. 2 describes the components of the oligonucleotides of
the present invention and demonstrates the ligation method.
[0018] FIG. 3: Protocol of Present Invention
[0019] FIG. 3 shows an embodiment of the method of the present
invention diagrammatically.
[0020] FIG. 4: Design of a gel free assay
[0021] FIG. 4 shows a second embodiment of the present invention as
a gel free detection assay. In this scheme, the different
oligonucleotide combinations will give different signals thus
indicating the allele content of the sample. P5 and ligated P5-PC
will not provide any signal.
[0022] FIG. 5: Case 1: genotyping a [CA].sub.13 repeat
[0023] FIG. 5 shows an example of the present invention for
genotyping a [CA].sub.13 repeat DNA.
[0024] FIG. 6: Case 2: genotyping a [CA].sub.14 repeat
[0025] FIG. 6 shows an example of the present invention for
genotyping a [CA].sub.14 repeat DNA.
[0026] FIG. 7: Results of genotyping [CA].sub.13 and [CA].sub.14
repeats
[0027] The top figure demonstrates the use of the present invention
in genotyping CA--repeated microsatellite; a [CA].sub.13 homozygous
(left); a [CA].sub.14 homozygous (middle) and a [CA].sub.13-14
heterozygous, (right), respectively. The bottom figure shows three
control lanes to demonstrate the specificity of the generated
ligation products: the reaction mixture contains all reagents
except the P3 oligonucleotide (left lane), no ligase (middle lane)
and no DNA template (right lane).
DEFINITIONS
[0028] Throughout the description of the present invention, several
terms are used that are specific to the technology of this field.
For the sake of clarity and to avoid any misunderstandings, these
definitions are provided:
[0029] Allele: At a given locus, a particular form of a gene or
genotype, specifying one of all the possible forms of the character
encoded by this locus. A diploid genome contains two alleles at any
given locus.
[0030] Discriminating Primers: A set of primers that are
individually specific for one allele, depending on the number of
repeat units that the primers possess at either their 3' or 5' end.
These primers differentiate between the different alleles.
[0031] Functional group: A moiety of chemical or proteinaeous
nature that is attached to either the 5' end or the 3' end of an
oligonucleotide allowing the latter to be purified by affinity.
[0032] Genotype: Set of alleles at a specified locus.
[0033] Hybridization: The process by which two nucleic acids are
linked together by base pairing, forming a duplex DNA of
complementary sequences. In this description, the hybridization
occurs between oligonucleotides and the PCR-amplified template. The
conditions of the assay are designed to reach a perfectly matched
duplex between the oligonucleotides and the template.
[0034] Label: An attachment linked to an oligonucleotide that
permits its specific detection via the signal emitted by the
attachment.
[0035] Ligase: An enzyme that catalyzes the formation of a
phosphodiester bond at the site of single-stranded break within a
DNA duplex.
[0036] Ligation: The process catalyzed by a DNA ligase.
[0037] Locus: A specified region of the genome.
[0038] Microsatellite: DNA of eukaryotic cells comprising highly
repetitive DNA sequences flanked by sequences unique to that locus.
In this description, microsatellite refers to mono-, di-, tri-,
tetra-, penta-, hexa-, hepta-, octa-, or nona-nucleotide repeated
regions.
[0039] Oligonucleotide: A short single-stranded deoxyribonucleic
acid molecule. In this description, the length of the
oligonucleotides varies from 5 bases to more than 32 bases.
[0040] Polymerase Chain Reaction (PCR) amplification: An enzymatic
process resulting in the exponential amplification of specific
region of a DNA template. The process uses a thermostable
polymerase, capable of replicating a DNA template from a primer. In
the presence of two primers, the region between them is amplified
following this process.
[0041] Thermostable ligase: A heat resistant enzyme capable of
performing ligase functions.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention relates to a method for genotyping
different alleles of microsatellite DNA by using a combination of
at least three oligonucleotides for each allele on the locus. In
particular, the present method comprises providing a sample
containing the microsatellite DNA; selecting combinations of at
least three oligonucleotides comprising a 5' primer (P5), wherein
the 5' primer comprises at least 5 base pairs complementary to the
3' flanking region of the repeated region of the microsatellite
DNA; a central primer (PC), wherein the central primer is
complementary to the repeated region of the microsatellite DNA; and
at plurality of 3' primers ([repeat-unit].sub.nP3 which comprises a
sequence that is complementary to the 5' flanking sequence of the
repeat region of the microsatellite, and which possesses at its 5'
end, a number (n=0, 1, 2 . . . ) of repeat units of the
microsatellite to genotype, in particular, cytosine-adenine,
(hereinafter, "[CA].sub.n") for a CA repeated DNA, mixing the
sample and primers such that the primers and microsatellite DNA
hybridize; adding a ligating reagent; and detecting the presence of
full ligation products that consist of the three oligonucleotide
primers linked together in a contiguous sequence. (see FIGS.
3-7).
[0043] The principle described in the present invention holds for
any changes in the orientation, length and modifications of the
oligonucleotides. One skilled in the art could easily design a
scheme in which the discriminating primers were the 5' primers,
P5[repeat-unit].sub.n. In particular, the present invention further
provides a plurality of 5' primers ([repeat-unit].sub.nP5) which
comprise a sequence that is complementary to the 3' flanking
sequence of the repeat region of the microsatellite, and which
possesses at its 3' end, a number (n=0, 1, 2 . . . ) of repeat
units of the microsatellite to genotype, in particular,
cytosine-adenine, (hereinafter, "[CA].sub.n") for a CA repeated
DNA; a central primer, PC, wherein the central primer is
complementary to the repeated region of the target strand of the
microsatellite DNA and contains a core sequence plus a varying
number of repeat units at its 3' end; and a 3' primer, P3, of a
fixed length, wherein P3 comprises at least 5 base pairs that are
complementary to the 5' flanking region of the repeated region of
the target microsatellite DNA strand.
[0044] The design of the oligonucleotides for the present invention
provides a low cost technology. In particular, the present
invention provides oligonucleotides wherein the 5' primer, P5, is
at least a 5-mer wherein a collection of 1024 oligonucleotides
(N.sup.5=A, C, G, or T) cover all possible sequences of this size.
The 3' primer can be a 5-mer or greater, preferably a 10 to 17-mer
(excluding the repeat units at its 5' end). The central primer or
repeat oligonucleotides can be used for the genotyping of many
microsatellites of the same repeat type, and a relatively small
collection of approximately 500 different oligonucleotides are
sufficient for genotyping of the vast majority of microsatellite
loci.
[0045] The present invention will not only increase the throughput
of the process at low cost, but it will also increase the precision
and accuracy of genotyping, that is expected to allow
cost-effective genotyping of pooled DNA samples. The present method
uses the capacity of the DNA ligase to join selectively designed
adjacent oligonucleotides that hybridize to a given DNA template.
(see FIG. 3). Combination of specifically designed oligonucleotides
for each allele in the marker in the assay will allows
determination of the allele content within the DNA sample.
[0046] Therefore, in one embodiment of the present invention, the
ligation reagent is T4 DNA ligase or a thermostable ligase.
However, one skilled in the art could use any ligase known in the
field to facilitate the ligation step.
[0047] The present invention uses combinations of three
oligonucleotides, P5, PC and P3, wherein the 5' primer, P5,
hybridizes to the complementary 3' flanking region of the
microsatellite, the central primer, PC, hybridizes to the
complementary repeated region of the microsatellite and the 3'
primers, [repeat-unit].sub.nP3, hybridize to the 5' complementary
flanking region of the microsatellite. (see FIG. 3).
[0048] In a preferred embodiment, P5 comprises a 5-mer sequence
complementary to the flanking region of the repeat and is
radiolabeled at its 5'-end. Therefore, in one embodiment, the
detecting step further comprises labeling the 5' end of the 5'
primer with a detectable label. PC comprises the complementary
sequence of the repeat and its length is dictated by the shortest
allele found at that particular microsatellite marker in the
population. The [repeat-unit].sub.nP3 is an ensemble of primers
with a core sequence complementary to the 5' flanking sequence of
the target strand and with a varying number (n=0, 1, 2, 3, . . . )
of repeat units at its 5' end. The number of repeat added at the 5'
end of [repeat-unit].sub.nP3 is for the genotyping of all the
existing alleles of the microsatellite marker.
[0049] The use of the present invention to perform genotyping with
pooled DNA will increase the value of the results of the assay.
Therefore, in one embodiment of the present invention, the sample
is a mixture of different samples containing a given microsatellite
locus from a number of individuals.
[0050] By way of example, the present invention demonstrates the
genotyping of the D6S471 locus which is a dinucleotide [CA]-repeat
microsatellite marker harbouring four different alleles,
[CA].sub.13, [CA].sub.14, [CA].sub.16 and [CA].sub.17. Dinucleotide
markers represent a degree of complexity, intrinsic to their
primary structure, suitable for the experimental development needed
to achieve both specificity and stability of DNA hybridization. The
methodology developed in genotyping this locus is applicable to
genotyping mono-, di-, tri-, tetra-, penta, hexa-, hepta-, octa-
and nona-nucleotide microsatellites. Therefore, in another
embodiment, the present invention, provides a method for genotyping
different microsatellite DNA at a locus by using a combination of
at least three oligonucleotides for each allele on the loci wherein
the sample of microsatellite DNA consist of mono-, di-, tri-,
tetra-, penta, hexa-, hepta-, octa- and nona-repeated alleles.
[0051] In yet another embodiment, the sample is an amplified PCR
fragment of the microsatellite DNA.
[0052] Other systems use only two oligonucleotides with one
ligation event which implies that for each microsatellite, there is
a need to synthesize long, specific oligonucleotide. In addition,
modified nucleosides are often used to improve stringency of the
hybridization step. These conditions increase the cost of large
scale genotyping projects. The present invention uses combinations
of three oligonucleotides, P5, PC and [repeat-unit].sub.n P3 and
two ligation to link the three oligonucleotides together which
improves the specificity and eliminates the need for long
oligonucleotides. In addition, specificity is achieved without
using modified nucleosides.
[0053] Following hybridization of the three oligonucleotides onto
the template, the DNA ligase joins them. After passing the ligation
products through a G-50 column, they are separated by gel
electrophoresis. Since P5 is radiolabelled at its 5' end (*), only
two ligation products will be visible upon exposure on a X-ray
film, P5-PC and P5-PC-P3. While the former is genotype-independent,
the latter will be formed exclusively when the particular
combination of oligonucleotides hybridize perfectly to a given
template, thus reflecting the genotype or allele content of the
locus. Therefore, in one embodiment of the present invention, the
detecting step comprises separating the ligation products according
to their size by gel electrophoresis.
[0054] However, a gel-free analysis system would provide automation
and efficiency. In another embodiment, the present invention
provides a method for detecting the ligation products by a gel free
system wherein the gel free system comprises covalently linking a
functional group to the 5' end of the 5'-primer, P5, wherein the
functional group exhibits specific binding to a component of a
solid support; and labeling the 3' end of the 3'-primers,
[repeat-unit].sub.nP3, with different detectable labels wherein the
different labels yield different signals. (see FIG. 4). The
different 3'-labels for each 3'-primer will differentiate and/or
discriminate between genotypes by revealing a different signal (or
signals) depending upon which oligonucleotide combination(s) will
ligate together. In one embodiment, the detectable labels are
fluorescent labels. In still another embodiment, the functional
group is biotin and the solid support is coated with
streptavidin.
[0055] In another embodiment, the present invention provides a
method for detecting the ligation products by a gel free system
wherein the gel free system comprises covalently linking a
functional group to the 3' end of the 3'-primer, P3, wherein the
functional group exhibits specific binding to a component of a
solid support; and labeling the 5' end of the 5'-primers,
[repeat-unit].sub.nP5 with different detectable labels wherein the
different labels yield different signals. The different 5'-labels
for each 5'-primer will differentiate and/or discriminate between
genotypes by revealing a different signal (or signals) depending
upon which oligonucleotide combination(s) will ligate together.
[0056] Therefore, the different labels attached to the
discriminating primers would be attached to the 5' end of
P5[repeat-unit]. In one embodiment, the detectable labels are
fluorescent labels. In still another embodiment, the functional
group is biotin and the solid support is coated with
streptavidin.
[0057] The present invention further provides a kit for detection
of the presence of different alleles at a locus by using a
combination of at least three oligonucleotides wherein the
oligonucleotides comprise sequences which are complementary to and
hybridize with one strand of the repeat region of a microsatellite
DNA and having terminal groups such that the oligonucleotides are
ligatable to each other when hybridized perfectly to the
microsatellite DNA.
[0058] In one embodiment of the present invention, a kit for the
detection of the presence of different microsatellite DNA at a
locus comprises combinations of at least three oligonulceotides and
instructions for use of the oligonucleotides in a suitable
container means, wherein the oligonucleotides comprise sequences
which are complementary to and hybridize with one strand of the
repeat region of a microsatellite DNA, wherein the oligonucleotides
comprise terminal groups such that the oligonucleotides are
ligatable to each other when hybridized to the microsatellite
DNA.
[0059] In another embodiment, the kit comprises a 5' primer; a
central primer; a plurality of 3' primers, and a ligating reagent.
In another embodiment, at least of one of the oligonucleotides is
labeled with a detectable label. In a preferred embodiment, the
label is a fluorescent label.
[0060] In yet another embodiment, the present invention provides a
kit for detection of the presence of different alleles at a locus
by separating the ligation products by a gel free system wherein
the kit comprises combinations of at least three oligonulceotides
and instructions for use of the oligonucleotides in a suitable
container means, wherein the oligonucleotides comprise at least one
primer that is covalently-linked to a functional group, wherein the
functional group exhibits specific binding to a component of a
solid support and at least one other primer is labeled with
different detectable labels, wherein the different labels yield
different signals.
[0061] In another embodiment, the kit comprises a 5' primer that is
covalently-linked to a functional group at its 5' end of the 5'
primer; a central primer; a plurality of 3' primers that are
labeled at their 3' end with different detectable labels wherein
the different labels yield different signals (discriminating
P3-primers); a ligating reagent, and a solid support. In a
preferred embodiment, the detectable labels are different
fluorescent labels. Still further, in another embodiment of the
present kit, the functional group is biotin and the solid support
is coated with streptavidin.
[0062] In another embodiment, the kit comprises a 3' primer that is
covalently-linked to a functional group at its 3' end of the 3'
primer; a central primer; a plurality of 5' primers that are
labeled at their 5' end with different detectable labels wherein
the different labels yield different signals (discriminating
P5-primers); a ligating reagent, and a solid support. In a
preferred embodiment, the detectable labels are different
fluorescent labels. Still further, in another embodiment of the
present kit, the functional group is biotin and the solid support
is coated with streptavidin.
EXAMPLES
[0063] These examples demonstrate the genotyping of homozygous and
heterozygous templates of 13 and 14 [CA] repeats. The
oligonucleotides were phosphorylated where the 5' primer, P5, was
radiolabeled. The target template was an enriched single stranded
DNA resulting from an asymmetric PCR reaction of the microsatellite
to genotype and was purified on a Sephadex column (G-50 micro spin,
Pharmacia). The DNA template and the oligonucleotides were first
heat-denatured (by transferring the respective tubes in boiling
water for three minute)s. The following components were mixed at
room temperature: five picomoles of each of the oligonucleotides, 3
microliters of formamide (final concentration of 30%), 2
microliters of 5X ligase buffer (Gibco/BRL) and 0.4 micrograms of
SSBP (Single Stranded Binding Protein, Promega). Then, 0.5
picomoles of the template was added to the mixture and 1
microliters of ligase (8 units, Gibco/BRL) was added immediately
after. After an incubation of 5 minutes at room temperature, the
reaction was stopped by heating (three minutes at 95.degree. C.).
The ligation products were then separated by gel electrophoresis in
a 12% denaturing polyacrylamide gel. Using these conditions,
templates of [CA].sub.13 versus [CA].sub.14 and vice versa, as well
as heterozygous templates of 13 and 14 CA (see FIG. 7) were
specifically genotyped. No modified nucleosides were used in these
assays.
[0064] When P5, PC and P3 (without any [CA].sub.0 unit at its 5'
end) were hybridized onto a [CA].sub.13 template, there were no
gaps or bulges due to unpairing bases and both junctions (P5-PC and
PC-P3) are ligated. The full discriminatory product was generated.
In contrast, when P5, PC and [CA].sub.1P3 are hybridized on a
[CA].sub.13 template, the [CA] unit of [CA].sub.1P3 bulged out,
thus preventing ligation. Note that the bulged [CA] was also found
at the P5-PC junction, but the ligated PC-[CA].sub.1P3 was not
visible upon exposure on an X-ray film.
Example I
Genotyping [CA].sub.13
[0065] For example, when genotyping a template of [CA].sub.13, only
the combination P5-PC-[CA].sub.0P3 ligated (see FIG. 4). The
combination P5-PC-[CA].sub.1P3 did not ligate on this template
because a [CA] unit bulged out at the junction of either P5-PC or
PC[CA]1P3 thus preventing ligation event to occur (see FIG. 4).
Example II
Genotyping [CA].sub.14
[0066] When genotyping a template of [CA].sub.14, the ligated
combination was P5-PC-[CA].sub.1 P3 (see FIG. 5). The combination
P5-PC-P3 did not ligate on a template of [CA].sub.14 because a gap
of dinucleotide [CA] existed between P5 and PC or between PC and P3
(see FIG. 5).
[0067] When P5-PC and [CA].sub.0P3 are hybridized onto a
[CA].sub.14 template, there was a gap of one [CA] unit at either
the P5-PC or PC-[CA].sub.0P3 junction and the full, discriminatory
product was not generated. However, P5, PC and [CA].sub.1P3
hybridize perfectly on the [CA].sub.14template and the full product
was generated.
Example III
Genotyping the D6S471 Locus
[0068] The genotype of the D6S471 locus was determined as an
example of the present invention. This locus was a dinucleotide
[CA]-repeat microsatellite marker harbouring four different
alleles, [CA].sub.13, [CA].sub.14, [CA].sub.16 and [CA].sub.17.
Dinucleotide markers represent a degree of complexity, intrinsic to
their primary structure, suitable for the experimental tuning
needed to achieve both specificity and stability of DNA
hybridization. The methodology developed in genotyping this locus
is thus applicable to mono-, di-, tri-, tetra, penta-, hexa-,
hepta-, octa- and nona-nucleotide microsatellites.
[0069] In the case of D6S471, PC consists of [CA].sub.13. The
various P3 oligonucleotides were of different lengths and consist
of a core sequence complementary to the 5'-repeat-adjacent
sequence, and of a number of n of [CA] units at its 5' end (where n
represents all numbers of dinucleotide necessary to fill the
sequence gap to PC, reflecting thus all alleles in the population
at that locus).
[0070] In the present model case, there were four different P3
reflecting the four alleles at the D6S471 locus. For example, when
genotyping a template of [CA].sub.13, only the combination P5-PC-P3
ligated (see FIG. 4). The combination P5-PC-[CA].sub.1P3 did not
ligate on this template because a [CA] unit bulged out at the
junction of either P5-PC or PC-[CA].sub.1P3 (see FIG. 4). When
genotyping a template of [CA].sub.14, the ligated combination was
P5-PC-[CA].sub.1P3 (see FIG. 5). The combination P5-PC-P3 did not
ligate on a template of [CA].sub.14 because a gap of dinucleotide
[CA] existed between P5 and PC or between PC and [CA].sub.0P3 (see
FIG. 5).
[0071] This application incorporates by reference the following
publications:
[0072] References
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[0094] WO 91/17239
[0095] WO 95/27078
[0096] WO 98/03673
[0097] EP 956359
* * * * *