US20050112657A1

US20050112657A1 - Methods and compositions for multiplex amplification of nucleic acids

Info

Publication number: US20050112657A1
Application number: US10/975,361
Authority: US
Inventors: Haiime Matsuzaki; Eric Murphy
Original assignee: Affymetrix Inc
Current assignee: Affymetrix Inc
Priority date: 1997-06-20
Filing date: 2004-10-29
Publication date: 2005-05-26
Also published as: AU7980298A; US6673579B2; US20020058281A1; WO1998059072A1; US6333179B1; US7053206B2; US20040126806A1

Abstract

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.

Description

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser. No. 60/050,405, filed on Jun. 20, 1997, the text of which is expressly incorporated herein.

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. 322:178 (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.
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:
C _A =C _L(L _A ÷L _L)²
C_Ais the concentration of primers for an amplicon A. C_Lis the concentration of primer for the longest amplicon. L_Ais the length of the amplicon A. L_Lis 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:
C _A =C _L(L _A ÷L _L)
wherein C_Ais the concentration of primers for an amplicon A; wherein C_Lis the concentration of primer for the longest amplicon; wherein L_Ais the length of the amplicon A; and wherein L_Lis 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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.
FIG. 2 illustrates the relationship for given values X and L_L, using the amplicons from different exons of the human p53 gene as an example.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that approximately equimolar yields of amplicons of varying lengths can be easily produced by multiplex PCR It has been determined that varying the primer concentrations as a function of the lengths of amplicons yields approximately equimolar amounts of amplicons of varying lengths. The relationship between primer concentration and the length of amplicons is as follows:
C _A =C _L(L _A /L _L)^X
wherein

- C_A=the concentration of primers for an amplicon A;
- C_L=the concentration of primer for the longest amplicon;
- L_A=the length of amplicon A;
- L_L=the length of the longest amplicon; and
- X is usually not zero and is often between one and three.

This relationship can be placed in a computer readable medium or be used with a computer system if desired.
FIG. 2 illustrates the relationship for given values X and L_L, using the amplicons from different exons of the human p53 gene as an example. Using primer concentrations as set forth, for example in FIG. 2, one skilled in the art can determine the optimum set of primer concentrations to yield approximately equimolar yields of varying length amplicons in a multiplex or multiple PCR. Preferably, primers having both comparable base composition and comparable melting temperatures are used. Also preferably, Mg⁺²concentration, annealing temperatures, and cycling times of the PCR are optimized prior to choosing the desired set of primer concentrations in accordance with the present invention.
PCR techniques applicable to the present invention include inter alia those discussed in PCR PRIMR: A LABORATORY Manual, Dieffenbac, C. W. and Dveksler, G. S., eds., Cold Spring Harbor Laboratory Press (1995).
The present application further provides primer sequences, primer concentrations, and experimental conditions useful in the amplification of the coding region of the human p53 gene. Particularly useful primers for amplification of exons of the p53 gene are set forth in Table 1.

Table 1

p53 Primer Set

20 primers in 1 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, sequences:


	Exon 2:	5′-TCATGCTGCATCCCCACTTTTCCTCTTG-3′
		5′TGGCCTGCCCTTCCAATGGATCCACTCA-3′

	Exon 3:	5′-AATTCATGGGACTGACTTTCTGCTCTTGTC.3′
		5′-TCCAGGTCCCAGCCCAACCCTTGTCC-3′

	Exon 4:	5′-GTCCTCTGACTGCTCTTTTCACCCATCTAC-3′
		5′-GGGATACGGCCAGGCATTGAAGTCTC-3′

	Exon 5:	5′-CTTGTGCCCTGACTTTCAACTCTGTCTC-3′
		5′-TGGGCAACCAGCCCTGTCGTCTCTCCA-3′

	Exon 6:	5′-CCAGGCCTCTGATTCCTCACTGATTGCTC-3′
		5′-GCCACTGACAACCACCCTTAACCCCTC-3′

	Exon 7:	5′-GCCTCATCTTGGGCCTGTGTTATCTCC-3′
		5′-GGCCAGTGTGCAGGGTGGCAAGTGGCTC-3′

	Exon 8:	5′-GTAGGACCTGATTTCCTTACTGCCTTGC-3′
		5′-ATAACTGCACCCTTGGTCTCCTCCACC-3′

	Exon 9:	5′-CACTTTTATCACCTTTCCTTGCCTCTTTCC-3′
		5′-AACTTTCCACTTGATAAGAGGTCCCAAGAC-3′

	Exon 10:	5′-ACTTACTTCTCCCCCTCCTGTGTTGCTGC-3′
		5′-ATGGAATCCTATGGCTTTCCAACCTAGGAAG-3′

	Exon 11:	5′-CATCTCTCCTCCCTGCTTCTGTCTCCTAC-3′
		5′-CTGACGCACACCTATTGCAAGCAAGQGTTC-3′

Table 2 shows particularly useful concentrations of the primers set forth in Table 1 for multiplex PCR amplification using the experimental conditions set forth in Table 3.

TABLE 2


Primer Concentrations in p53 Primer Set

	Amplicon	Length	Primer Concs

Longest	4	368 bp	450.0 nM
	5	272 bp	245.8 nM
	8	241 bp	193.0 nM
	11	231 bp	177.3 nM
	10	210 bp	146.5 nM
	6	204 bp	138.3 nM
	7	175 bp	101.8 nM
	2	164 bp	89.4 nM
	9	146 bp	70.8 nM
Shortest	3	90 bp	26.9 nM

Values of X 2
Typical values of C_L450 nM

TABLE 3


Multiplex PCR

Start with 250 ng of Template DNA.
PCR Components for 100 ul PCR in 0.2 ml thin walled tubes:

Stock
Conc	Final Conc	for 1 reaction

Buffer (No Mg)	10	X	1	X	10.0 ul
MgCl₂	25	mM	2.5	mM	10.0 ul
dATP
	10	mM	200	uM	2.0 ul
dCTP
	10	mM	200	uM	2.0 ul
dGTP
	10	mM	200	uM	2.0 ul
dTTP
	10	mM	200	uM	2.0 ul
Taq GOLD	5	U/ul	10	U	2.0 ul
p53 Primer Set	20	X	1	X	5.0 ul
Water
Human genomic
			250	ng
DNA

	Total	100.0 ul
	Volume

Final Concentrations in Buffer (No Mg) are 10 mM Tris-HCl (pH 8.3),

50 mM KCl

Taq GOLD is AmpliTaq Gold ™ from Perkin Elmer catalog # N808-0243

PCR Cycles:

35 Cycles:	94 C.	10 min
	94 C.	30 sec
	60 C.	30 sec
	72 C.	45 sec
	72 C.	10 min

To visualize amplicons by gel Analysis:

Visualize PCR products on 4% NuSieve Agarose Gel

NuSieve ™ Agarose 3:1 is from FMC catalog # 50092

Load 5 ul of PCR + loading buffer

Use

50 bp Ladder (Gibco/BRL catalog # 10416-014) as size marker

Run gel at 125 Volts for 30 min. to 90 min.

	Expected
	PCR Products:		Order in Gel:

	Amplicon	Length	Amplicon	Length

	Exon
2	164 bp	Exon	4	368 bp
	Exon
3	90 bp	Exon	5	272 bp
	Exon
4	368 bp	Exon	8	241 bp
	Exon
5	272 bp	Exon	11	225 bp
	Exon
6	204 bp	Exon	10	210 bp
	Exon 7	175 bp	Exon	6	204 bp
	Exon
8	241 bp	Exon 7	175 bp
	Exon 9	146 bp	Exon	2	164 bp
	Exon
10	210 bp	Exon 9	146 bp
	Exon
11	225 bp	Exon	3	90 bp

Using the methods and reagents provided herein, we achieved multiplex PCR amplification of coding regions shown of the human p53 gene in approximately equimolar amounts. That desirable result was achieved in a single-tube reaction. The achievement of such desirable results with the remarkable convenience of a single tube reaction further illustrates the contribution to the art made by the present invention.
The methods and compositions of the present invention are useful in virtually any context in which equimolar yields of various PCR products are desired. Such contexts include without limitation paternity testing, forensic analysis, genetic screening, polymorphism detection, and mutation analyses. The present invention can be used to amplify nucleic acids for all forms of sequence analysis known to those skilled in the art. Sequence analysis techniques includes, for example, dideoxy-sequencing and sequence analysis using high-density nucleic acid arrays: the GeneChip® probe arrays or VLSIPS™ technology of Affymetrix, Inc. High density nucleic acid arrays are discussed for example in Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler, J., Lockhart, D. J., Morris, M. S., & Fodor, S. P., Science 5287, 610-614 (1996), U.S. Pat. No. 5,445,934, and International Publication No. WO 95/11995 corresponding to PCT Application No. PCT/US94/12305.
The p53 gene and its protein product are discussed in Molecular Biology of the Cell, 3rd Edition, Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D., Garland Publishing (1994) at pages 889 and 1284-1289.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of any appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method of performing multiple polymerase chain reactions in a single vessel, comprising:

priming DNA synthesis on a template in a vessel with at least two sets of primers, wherein each of the sets of primers in the vessel is at a predetermined ratio, wherein the ratio is described by:

C _A =C _L(L _A ÷L _L)²

wherein C_Ais the concentration of primers for an amplicon A; wherein C_Lis the concentration of primer for the longest amplicon; wherein L_Ais the length of the amplicon A; and wherein L_Lis the length of the longest amplicon.

2. The method of claim 1 wherein the template is genomic DNA encoding p53.

3. The method of claim 1 wherein the template is a cDNA encoding p53.

4. The method of claim 1 wherein the primers amplify at least 2 exons of p53 selected from the group consisting of exons 2-11.

5. The method of claim 1 wherein the primers amplify at least 4 exons of p53 selected from the group consisting of exons 2-11.

6. The method of claim 1 wherein the primers amplify exons 2-11 of p53.

7. The method of claim 4 wherein the primers are selected from those shown in SEQ ID NO: 1-20.

8. The method of claim 5 wherein the primers are selected from those shown in SEQ ID NO: 1-20.

9. The method of claim 6 wherein the primers are shown in SEQ ID NOS: 1-20.

10. The method of claim 9 wherein the primers are present in the following ratios: 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).

11-24. (canceled)