Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-04-14
2000-08-29
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, C12P 1934, C12Q 168
Patent
active
061107107
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The impact of the polymerase chain reaction (PCR) on biological research is perhaps most clearly evidenced by the number and variety of specific applications of this technique. The power and versatility of PCR amplification has transformed this method into a standard tool in molecular biology research and created an ever increasing demand for PCR instrumentation and reagents. An unfortunate corollary of PCR is that errors resulting from properties inherent to the reagents are amplified and unwittingly interpreted as the target sequence.
One prevalent source of error is DNA polymerase catalyzed non-templated addition of a nucleotide to the 3' hydroxyl terminus of duplex PCR products. This activity has been reported for DNA polymerases from Thermus aquaticus, polymerase .alpha. from chick embryo, rat polymerase .beta., reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae. Clark, Nucleic Acids Research, 16(20):9677-9686 (1988). In the presence of all four deoxynucleotidetriphosphates (dNTPs), these DNA polymerases differ in the efficiency with which particular dNTPs are added but generally display a preference for non-templated addition of dATP. Clark reported that template independent addition required a duplex DNA substrate but stated that adenylation cannot involve the use of coding information from the template strand. More recently, it was reported that Taq polymerase is relatively resistant to adding an extra nucleotide on a 3' terminal adenosine, and relatively efficient at adding an adenosine residue on a 3' terminal cytosine. Hu, DNA and Cell Biology, 12(8):763-770 (1993).
At best, non-templated 3' adenylation of an otherwise blunt-ended duplex may result in inefficient cloning. More disturbing, however, is its impact on genotyping, where artifactual variations in marker size may adversely impact interpretations of family relationships, medical diagnosis, and forensics. Moreover, full automation of genotyping has been hampered by the necessity of manually editing collected data to correct for allele misidentification due to the unpredictability of non-templated nucleotide addition.
What is needed in the art is a means to control (i.e., reduce or more consistently promote) non-templated nucleotide addition. Further, a method to facilitate template independent addition is needed to exploit the advantageous properties of 3' adenylated PCR products. Quite surprisingly, the subject invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention relates to a method of promoting a non-templated 3' adenosine addition to a PCR amplification product. The method comprises the steps of amplifying a target with a primer comprising the 5' terminal sequence 5'-G-T-K-N-3' wherein K is G or T/U, N is A, C, G, or T/U and wherein at least one of said terminal sequence residues is not complementary to the target sequence. In one embodiment, the 5' terminal sequence is 5'-G-T-K-N-V-N-N-3' wherein V is A, C, or G. In another embodiment, the 5' terminal sequence is 5'-G-T-K-T-V-N-N-3'. In a preferred embodiment, the 5' terminal sequence is 5'-G-T-T-T-V-N-N-3'. Typically, adenosine addition is catalyzed by a thermostable DNA polymerase such as Taq polymerase.
In another aspect, the present invention relates to a method of resisting non-templated 3' adenosine addition to a PCR amplification product. The method comprises the steps of amplifying a target with a primer comprising the 5' terminal sequence 5'-Y-M-V-3' wherein Y is C or T/U, M is A or C, V is A, C, or G, and wherein at least one of said terminal sequence residues is not complementary to the target strand. In a preferred embodiment, the 5' terminal sequence is 5'-Y-M-V-V-N-N-N-3' wherein N is A, C, G, or T/U. In preferred embodiments, the primer is linked at the 5' terminus to an adenylation inhibiting group such as biotin or primary amine.
In a further aspect of the present invention, primers comprising 5' terminal sequences for promoting or resisting
REFERENCES:
patent: 4683195 (1987-07-01), Mullis et al.
patent: 5008182 (1991-04-01), Sninsky et al.
patent: 5411876 (1995-05-01), Bloch et al.
patent: 5451505 (1995-09-01), Dollinger
Clark et al. Nucleic Acid Res. 16(20):9677-9686, Nov. 1988.
Gengxi Hu, DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment, DNA and Cell Biology 12(8):763-770 (Oct. 1993).
Jeffrey R. Smith , et al., "Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase," Genome Research, pp. 312-317, (Jul. 18, 1995).
Michael J. Brownstein, et al., "Short technical reports," BioTechniques, 20(6):1004-1010.
Brownstein Michael J.
Carpten John D.
Smith Jeffrey R.
Campbell Eggerton A.
The United States of America as represented by the Secretary of
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