Amplification of GC-rich DNA for high-throughput family-based genetic studies.
Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80 % GC contents and a size of >300 bp. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65-85 % and sizes up to 870 bp. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.
School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore, Pakistan. naz.sbs@pu.edu.pk
Sadaf
S
Naz
Amara
A
Fatima
D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D020862:GC Rich Sequence; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008623:Mercaptoethanol / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D011506:Proteins; C548551:TPRN protein, human; D008623:Mercaptoethanol; D004247:DNA; D004259:DNA-Directed DNA Polymerase