Method of primer-extension preamplification PCR

Details Method of primer-extension preamplification PCR Method of primer-extension preamplification PCR

1999-03-16

2002-04-02

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S091200, C435S006120

Reexamination Certificate

active

06365375

ABSTRACT:

The present application claims priority to German Patent Application No. 198 13 317.0, filed Mar. 26, 1998.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention provides an improved method of “whole genome amplification” (WGA) that is suitable for performing DNA analysis starting with just one or a few cells. The improvement of the method basically lies in the fact that, to amplify the DNA, a mixture of two DNA polymerases is used, at least one of which possesses 3′-5′ exonuclease activity.
2. Description of Related Art
WGA methods are especially important in the field of differential tumor diagnostics. The goal of differential tumor diagnostics at the molecular level is to analyze nucleic acid samples from single cells or cell populations that contain no non-malignant cells (Emmert-Buck et al., 1996; Böhm and Wielang, 1997). To obtain the appropriate samples, therefore, cell sorting methods (Abeln et al., 1994, Barret et al., 1996), microdissection methods (Shibata et al., 1992, Shibata et al., 1993, Emmert-Buck et al., 1994, Noguchi et al., 1994, Zhuang et al., 1995, Böhm et al., 1997), and laser microdissection (Schütze and Clement-Sengewald, 1994) are used with increasing frequency.
Especially sensitive methods of nucleic acid amplification are required before a molecular analysis of single cells or populations of a few cells can be performed, however. According to the prior art, methods of “whole genome amplification” (WGA) are especially well-suited for this application. These are methods that comprise two consecutive amplification reactions. The first amplification reaction is carried out using randomized primers, and the second amplification is carried out using specific primers. WGA can be used, for instance, to diagnose hereditary diseases as part of pre-implantation diagnostic testing of biopsied blastomere cells (Kristianson et al., 1994, Snabes et al., 1994, van der Veyer et al., 1995), or as part of prenatal diagnostic testing of nucleated erythrocytes in maternal blood (Sekizawa et al., 1996).
According to the prior art, WGA is usually used to analyze microsatellites in tumor biopsies to detect microsatellite instability or the loss of heterozygosity. According to the prior art, the analyzed sample must contain so many cells, however, that a quantitatively disproportionate amplification of individual alleles caused by accidental preparation artifacts is ruled out (Zhang et al., 1992, Barret et al., 1995, Cheung and Nelson, 1996, Faulkner and Leigh, 1998). For instance, a batch of about 1,000 cells was investigated in a microsatellite analysis of FACS-sorted aneuploidal esophageal tumor cells (Barret et al., 1995).
In contrast to conventional in situ hybridization (Van Ommen et al., 1995) or conventional, specific PCR (Becker et al., 1996), multiple analyses of the same sample can be carried out using WGA methods. Two different methods are used. In “degenerate oligonucleotide primer PCR” (DOP-PCR), amplification primers with defined sequences on the 5′ and 3′ ends and a randomized hexamer region in the middle of the primer are used (Telenius et al., 1992). Starting with only slightly stringent conditions in the first 5 thermal cycles, the next 35 thermal cycles are carried out under more stringent conditions at a higher annealing temperature so that, during these cycles, only completely complementary primers can bind to the DNA to be amplified. These methods are used, for instance, as the first step before performing an in situ hybridization with flow-sorted chromosomes (Blennow et al., 1992; Telenius et al., 1992; Kallionemie et al., 1994), or to perform comparative genomic hybridization (CGH) (Du-Manoir et al., 1993; Schlegel et al., 1995).
An alternative principle of WGA is “primer-extension preamplification” (PEP-PCR, Zhang et al., 1992). In contrast to the DOP-PCR, this method uses completely randomized 15-mer amplification primers. During 50 consecutive thermal cycles, denaturing is first carried out at 92° C., followed by hybridization under only slightly stringent temperature conditions at 37° C. This temperature is increased successively to 55° C. at a rate of about 0.1° C./second. At this temperature, the polymerase extension reaction takes place for another 4 minutes.
All methods known in the prior art (von Eggeling and Spielvogel, 1995) have the disadvantage of insufficient sensitivity, however, because a relatively large number of cells must be used to increase the possibility of obtaining an amplification product. In addition, the sensitivity of the assay is reduced even more as the length of the fragment to be amplified increases. For this reason, the methods known in the prior art had only been used in the amplification of relatively small fragments with a length of up to 580 base pairs (Snabes et al., 1994).
Another main disadvantage of the PEP-PCR known in the prior art lies in the fact that a convincing DNA mutation analysis has never been reliably carried out due to the inherent error rate of the Taq polymerase used. The error rate is due to the fact that using Taq polymerase during amplification leads to AT/GC transitions in the amplification product (Keohvong and Thily, 1989). In addition, deletion mutations may arise when Taq polymerase is used if the DNA to be amplified is capable of forming secondary structures (Carriello et al., 1991). The risk of obtaining amplification artifacts is especially high with WGA, however, because more than 80 amplification cycles are usually carried out during the 2 to 3 amplification reactions.
To avoid sequence artifacts during nucleic acid amplification, the use of DNA polymerases with 3′-5′ exonuclease activity was also known in the prior art (Flaman et al., 1994; Casas and Kirkpontrick, 1996). The use of polymerases without 3′-5′ exonuclease activity for WGA, however, leads to a further reduction in the sensitivity of the method, because such polymerases possess much less processivity than Taq DNA polymerases, for instance. As a result, the products created during preamplification when randomized primers are used are not long enough to serve as matrices for the subsequent specific PCR reaction if the fragment to be amplified exceeds a certain size.
The technical object to be solved with this invention was therefore to develop a method with which, starting with the smallest possible number of cells, specific nucleic acid fragments of high quality, i.e., containing no sequence artifacts, could be amplified and then analyzed. The quality of the amplification products should make it possible to carry out reliable mutation analyses, sequence analyses, and unequivocally interpretable microsatellite analyses. This objective is solved by an improved method of primer-extension preamplification (PEP-PCR, Zhang et al., 1992).
SUMMARY OF THE INVENTION
Object of the invention is therefore a method for the amplification of nucleic acid fragments from a sample comprising two or three thermocyclic amplification reactions in which completely randomized primers are used in the first amplification reaction and specific primers are used in the second amplification reaction, characterized in that, to amplify the DNA, a mixture of at least two DNA polymerases is used, at least one of which possesses 3′-5′ exonuclease activity. This characteristic is also called proofreading activity in the context of polymerases (Flaman et al., 1994).
An amplification reaction comprises about 20 to 60 thermal cycles. The first amplification reaction preferably comprises at least 40 thermal cycles and, most preferably, at least 50 thermal cycles. The second amplification reaction preferably comprises at least 30 thermal cycles, and most preferably, at least 40 thermal cycles.
Each thermal cycle comprises a denaturing phase, an annealing phase, and at least one elongation phase. Denaturation into single strands preferably takes place at temperatures of between 90° C. and 96° C. The annealing phase to hybridize the primers with the target nucleic acid preferably takes p

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