Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-11-21
2004-01-06
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06673579
ABSTRACT:
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.
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:
C
A
=C
L
(
L
A
÷L
L
)
2
C
A
is the concentration of primers for an amplicon A. C
L
is the concentration of primer for the longest amplicon. L
A
is the length of the amplicon A. L
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): exon4 (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 NOS: 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
)
2
wherein C
A
is the concentration of primers for an amplicon A; wherein C
L
is the concentration of primer for the longest amplicon; wherein L
A
is the length of the amplicon A; and wherein L
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.
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patent: 5700637 (1997-12-01), Southern
patent: 5705365 (1998-01-01), Ryder et al.
patent: 6333179 (2001-12-01), Matsuzaki et al.
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patent: WO 89/10977 (1989-11-01), None
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patent: WO 96/10648 (1996-04-01), None
patent: WO 96/39535 (1996-12-01), None
patent: WO 96/41012 (1996-12-01), None
Haff, Lawrence A., “Improved Quantitative PCR Using Nested Primers”, PCR Methods and Applications 3:332-337.
Matsuzaki Hajime
Murphy Eric
Affymetrix Inc.
Banner & Witcoff , Ltd.
Lu Frank Wei Min
Whisenant Ethan
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