Optimization of DNA amplification reactions
The present invention relates to methods and uses where a combination of (i) an enzyme exhibiting 5′→3′ exonuclease activity and specifically degrading 5′-phosphorylated strands of double-stranded nucleic acids is used, and (ii) one or more primer pair(s), each primer being 5′ phosphorylated, in a method comprising at least a first and a second amplification reaction of a first and a second template, respectively, for preventing contamination of the second amplification reaction with amplification products of the first amplification reaction, as well as novel methods and kits using the combination. Also, the present invention relates to kits comprising the enzyme and either 5′-phosphorylated primers or a polynucleotide kinase.
1. A method for generating nucleic acid amplicons, the method comprising:
a) bringing a first DNA template into contact with nucleotides, a DNA polymerase and primers, wherein at least one of the primers is phosphorylated at its 5′-end;
b) amplifying, in a first amplification reaction, the first DNA template, and thereby generating double-stranded amplification products with 5′-phosphorylated ends;
c) bringing double-stranded amplification products from step b) or a reaction mixture containing the double-stranded amplification products from step b) with an enzyme exhibiting 5′→3′ exonuclease activity and specifically degrading 5′-phosphorylated strands of the double-stranded amplification products for a time period and under conditions that enable the degradation of the first amplification reaction; and
d) performing at least one other amplification reaction with a second DNA template, thus generating nucleic acid amplicons.
2. The method of claim 1 , further comprising performing a DNA library amplification step to generate DNA sequencing libraries.
3. The method of claim 1 , wherein in step a) the first DNA template is a first next generation sequencing (NGS) library, in step b) the first amplification reaction is a next generation sequencing (NGS) library amplification reaction, and in step d) the second DNA template is a second NGS library and the at least one other amplification reaction is a second NGS library amplification reaction.
4. The method of claim 1 , further comprising performing a clonal amplification step for generating next generation sequencing (NGS) templates.
5. The method of claim 1 , wherein in step a) the first DNA template is a first DNA library, in step b) the first amplification reaction is a clonal amplification reaction, and in step d) the second DNA template is a second DNA library and the at least one other amplification reaction is a second clonal amplification reaction.
6. The method of claim 1 , further comprising performing a step of multiplex PCR or long-range PCR for NGS target enrichment.
7. The method of claim 1 , wherein in step b) the first amplification reaction is a first multiplex or long-range polymerase chain reaction (PCR) of a first DNA template, thus generating an amplified target region of interest, and that in step d) the at least one other amplification reaction is a second multiplex PCR amplification reaction of a second DNA template.
8. The method of claim 1 , wherein the enzyme exhibiting 5′→3′ exonuclease activity and specifically degrading 5′-phosphorylated strands of double-stranded nucleic acids is lambda exonuclease.
9. The method of claim 1 further comprising preventing contamination of the second amplification reaction with amplification products of the first amplification reaction by using one or more multiple primer pair(s) in the first amplification reaction, at least one of the primers being 5′ phosphorylated and then prior to the start of the second amplification reaction incubating the second reaction mixture with an enzyme exhibiting 5′→3′ exonuclease activity and specifically degrading 5′ phosphorylated strands of double stranded nucleic acids.
10. The method of claim 9 , wherein the enzyme exhibiting 5′→3′ exonuclease activity and specifically degrading 5′-phosphorylated strands of double-stranded nucleic acids is lambda exonuclease.