Methods and compositions for multiplex amplification of nucleic acids

A method is described for predetermining ratios of primer pairs present in a single reaction vessel so as to achieve approximately equimolar yield of products. The ratios are determined as a function of the length of the amplicon and the length of other amplicons being simultaneously tested. The primers may desirably be for p53 gene sequences.

BACKGROUND OF THE INVENTION
 The polymerase chain reaction (PCR) is a simple and versatile method to
 amplify in vitro a specific segment of DNA for subsequent study (Saiki et
 al., Science 230: 1350 (1985); Saiki et al., Science 235:487 (1985)). The
 PCR method has gained widespread use in biomedical research, and has
 revolutionized the accurate and early diagnosis of many inherited and
 acquired genetic disorders (Eisenstein, N. Engl. J. Med. 332:178 (1990)),
 particularly those caused by point mutations or small insertions or
 deletions including sickle cell anemia (Saiki et al., Science 230:1350
 (1985)), hemophilia A (Kogan et al., N. EngL. J. Med. 317:985 (1987)),
 Tay-Sach's disease (Myerowitz, Proc. Natl. Acad. Sci. USA 85:3955 (1988);
 Myerowitz et al., J. Biol. Chem. 263:18587 (1988)), cystic fibrosis
 (Riordan et al., Science 245:1066 (1989)), and many others. With PCR, it
 is also possible to detect heterozygotic carriers in recessive disorders.
 Polymerase chain reaction (PCR) is used for a variety of purposes. PCR can
 be used to amplify genomic DNA or other sources of nucleic acids for
 analysis. It is often desirable to be able to achieve equimolar yields of
 different length amplicons when performing multiplex PCR or multiple PCR
 reactions. Having an approximately equimolar yield of amplicons is
 particularly useful, for example, when approximately equal representation
 of certain regions of genomic DNA amplified after multiplex PCR is
 desired. Prior to the methods of present invention, finding the
 appropriate experimental conditions useful to achieve this result has been
 difficult because PCR amplifies nucleic acids having different lengths
 with different efficiencies. The yield of longer amplicons is often less
 than the yield of shorter amplicons because of those differences in PCR
 amplification efficiency. FIG. 1 shows the difference in yields that one
 might expect, for example, when starting with equal primer concentrations
 used to amplify amplicons of varying lengths: A, B, C. There is a
 continuing need in the art for methods which permit the amplification of
 different sequences with the same efficiency so that approximately
 equimolar products result.
 SUMMARY OF THE INVENTION
 It is an object of the present invention to provide a method of performing
 multiplex PCR which achieve approximately equimolar products.
 It is another object of the invention to provide a set of primers for
 amplification of p53.
 It is yet another object of the invention to provide a set of primers for
 amplification of p53 to achieve approximately equimolar products.
 It is still another object of the invention to provide a mixture of primers
 for performing multiplex PCR.
 These and other objects of the invention are provided by one or more of the
 embodiments provided below. In one embodiment of the invention a method of
 performing multiple polymerase chain reactions in a single vessel is
 provided. The method comprises the steps of priming DNA synthesis on a
 template in a vessel with at least two sets of primers. The primers are
 present in the vessel at a predetermined ratio which is described by the
 formula:
EQU C.sub.A =C.sub.L (L.sub.A.div.L.sub.L).sup.2
 C.sub.A is the concentration of primers for an amplicon A. C.sub.L is the
 concentration of primer for the longest amplicon. L.sub.A is the length of
 the amplicon A. L.sub.L is the length of the longest amplicon.
 Another embodiment provided by the invention is a method of performing
 multiple polymerase chain reactions in a single vessel. The method
 comprises priming DNA synthesis on a genomic p53 template in a vessel with
 ten sets of primers which amplify exons 2-11 of p53. The primers are shown
 in SEQ ID NO: ID NOS: 1-20. The primers are present in the vessel at the
 following ratio: exon 2 (89.4): exon 3 (26.9): exon 4 (450): exon 5
 (245.8): exon 6 (138.3): exon 7 (101.8): exon 8 (193.0): exon 9 (70.8):
 exon 10 (146.5): exon 11 (177.3).
 According to still another embodiment of the invention a set of primers for
 performing multiple polymerase chain reactions in a single vessel is
 provided. The set comprises twenty primers having sequences as shown in
 SEQ ID NO: 1-20.
 According to yet another embodiment of the invention a mixture of primers
 for performing multiplex polymerase chain reaction is provided. The
 primers are present in the mixture at a predetermined ratio to each other.
 The ratio of the concentrations of the primers is described by:
EQU C.sub.A =C.sub.L (L.sub.A.div.L.sub.L).sup.2
 wherein C.sub.A is the concentration of primers for an amplicon A; wherein
 C.sub.L is the concentration of primer for the longest amplicon; wherein
 L.sub.A is the length of the amplicon A; and wherein L.sub.L is the length
 of the longest amplicon.
 The present invention thus provides the art with a method useful for
 performing multiplex PCR. This method is particularly useful for
 amplification of multiple exons of p53. Moreover, a particular primer set
 useful for performing such multiplex PCR is also provided.