Universal primer sequence for multiplex DNA amplification

Details Universal primer sequence for multiplex DNA amplification Universal primer sequence for multiplex DNA amplification

1998-02-10

2001-03-27

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S024300

Reexamination Certificate

active

06207372

ABSTRACT:

FIELD OF THE INVENTION
This invention pertains to universal primers having use in amplification of DNA sequences by methods such as polymerase chain reaction (PCR), specifically to primers that allow the simultaneous amplification of a multiplicity of DNA sequences.
BACKGROUND OF THE INVENTION
Polymerase chain reaction (PCR) is a method whereby virtually any DNA sequence can be selectively amplified. The method involves using paired sets of oligonucleotides of predetermined sequence that hybridize to opposite strands of DNA and define the limits of the sequence to be amplified. The oligonucleotides prime multiple sequential rounds of DNA synthesis catalyzed by a thermostable DNA polymerase. Each round of synthesis is typically separated by a melting and re-annealing step, allowing a given DNA sequence to be amplified several hundred-fold in less than an hour (Saiki et al.,
Science
239:487, 1988).
With the rapid advances in mammalian molecular genetics, an ever increasing number of disease genes have been identified. Accordingly, PCR has gained widespread use for the diagnosis of inherited disorders and the susceptibility to disease. Typically, the region of interest is amplified from either genomic DNA or from a source of specific cDNA encoding the cognate gene product. Mutations or polymorphisms are then identified by subjecting the amplified DNA to analytical techniques such as DNA sequencing, hybridization with allele-specific oligonucleotides (ASOs), oligonucleotide ligation, restriction endonuclease cleavage or single-strand conformational polymorphism (SSCP) analysis.
For the analysis of small genes and transcripts, or genes where the mutant allele or polymorphism is well characterized, amplification of a single defined region of DNA is sometimes sufficient. When analyzing large genes and transcripts or undefined genes, however, multiple individual PCR reactions are often required to identify critical base changes or deletions. Thus, to streamline the analysis of large complex genes, multiplex PCR (i.e., the simultaneous amplification of different target DNA sequences in a single PCR reaction) has been utilized.
The results obtained with multiplex PCR are, however, frequently complicated by artifacts of the amplification procedure. These include “false-negative” results due to reaction failure and “false-positive” results such as the amplification of spurious products, which may be caused by annealing of the primers to sequences which are related to, but distinct from, the true recognition sequences.
For use in multiplex PCR, a primer should be designed so that its predicted hybridization kinetics are similar to those of the other primers used in the same multiplex reaction. While the annealing temperatures and primer concentrations may be calculated to some degree, conditions generally have to be empirically determined for each multiplex reaction. Since the possibility of non-specific priming increases with each additional primer pair, conditions must be modified as necessary as individual primer sets are added. Moreover, artifacts that result from competition for resources (e.g., depletion of primers) are augmented in multiplex PCR, since differences in the yields of unequally amplified fragments are enhanced with each cycle.
Weighardt et al. (
PCR Meth.App.
3:77, 1993) describe the use of 5′-tailed oligonucleotides for PCR. However, a key feature of this amplification method involves separate annealing and primer extension reactions for each primer, which is not practical in a multiplex context. Therefore, complete optimization of the reaction conditions for multiplex PCR can become labor intensive and time consuming. Since different multiplex PCRs may each have unique reaction conditions, development of new diagnostic tests can become very costly.
Thus, there is a need in the art for primers that allow multiplex PCR reactions to be designed and carried out without elaborate optimization steps, irrespective of the potentially divergent properties of the different primers used. Furthermore, there is a need in the art for primers that allow multiplex PCR reactions that, under the same reaction conditions, simultaneously produce equivalent amounts of each of many amplification products.
SUMMARY OF THE INVENTION
This invention pertains to primers that allow simultaneous amplification of multiple DNA target sequences present in a DNA sample. According to the invention, the DNA sample in a single reaction mixture is contacted with a multiplicity of paired oligonucleotide primers having the structure 5′-XY-3′, wherein: X comprises a sequence that does not hybridize to the target sequence; the melting temperature of a hybrid between X and its complement in the absence of other sequences is greater than about 60° C.; and Y comprises a sequence contained within or flanking the target sequence or its complement.
Multiple cycles of melting, reannealing, and DNA synthesis (i.e., a PCR reaction) are thereafter performed with the above mentioned DNA sample and invention oligonucleotide primers. Amplified target sequences may then be detected by any method, including, for example, hybridization with allele-specific oligonucleotides, restriction endonuclease cleavage, or single-strand conformational polymorphism (SSCP) analysis.
The invention also encompasses a method for detecting multiple defined target DNA sequences in a DNA sample. This method is carried out by performing the same procedure set forth above, in which the 3′ sequence of one primer of each pair comprises a target DNA sequence itself or its complement. The method includes a further step of detecting the amplification products, preferably by gel electrophoresis. In this embodiment, the presence or absence of an amplification product is diagnostic of the presence or absence of the target sequence in the original DNA sample.
In another aspect, the invention encompasses methods for high-throughput genetic screening. The method, which allows the rapid and simultaneous detection of multiple defined target DNA sequences in DNA samples obtained from a multiplicity of individuals, is carried out by simultaneously amplifying many different target sequences from a large number of patient DNA samples, using oligonucleotide primer pairs as above.
In yet another aspect, the present invention provides single-stranded oligonucleotide DNA primers for amplification of a target DNA sequence in a multiplex polymerase chain reaction. The primers have the structure 35 5′-XY-3′, wherein X comprises an invention primer sequence, and Y comprises a sequence contained within or flanking a target sequence or its complement. Typically, Y comprises a sequence from 17 to 25 bases in length, and the melting temperature of hybrids between the primers and their complements is at least 72° C.
The methods and compositions of the present invention can be applied to the diagnosis of genetic and infectious diseases, gender determination, genetic linkage analysis, and forensic studies.


REFERENCES:
patent: 5104792 (1992-04-01), Silver et al.
patent: 5474896 (1995-12-01), Dujon et al.
patent: 5518901 (1996-05-01), Murtagh
patent: 5882856 (1999-03-01), Shuber
patent: 628640 (1994-12-01), None
patent: 89/12684 (1989-12-01), None
patent: WO 93/18177 (1993-09-01), None
patent: WO 93/18178 (1993-09-01), None
Edwards et al. Multiplex PCR: advantages, development, and applications, PCR Methods and Applications, vol. 3, pp. S65-S75, 1994.*
Berg et al. (1994)Biotechniques, 17(5):896-901.
Chenhui, T. (1993)The Journal of Heredity, 84(3):236-237.
Chetverin, A. (1994)Bio/Technology, 12:1093-1099.
Ebrahimi et al. (1992)J. Chem. Soc. Chem. Commun., 1398-1399.
Edwards et al, (1994)PCR Methods and Applications, 3:565-575.
Feng et al. (1991)Physical Review B, 43(11):9284-9286.
Feng et al. (1992) “Modified Self-Consistent Phonon Calculation Of The Dependence Of DNA Melting Temperature On Guanine-Cytosine Content” 46(12):8002-8006.
Guatelli et al. (1989)Clinical Microbiology Reviews, 2(2):217-226.
Hung et al. (1990)Nucleic Acids Researc

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