Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with Taq DNA polymerase.
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PMID:Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates. 3071 22

Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5-10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.
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PMID:The impact of common PCR inhibitors on forensic MPS analysis. 3087 22


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