PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping

Abstract

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

“…The miscoding error percentage reaches an average of 0.36%, which is more than twice higher than for molecules with one native N strand ( Figure 3 ). These miscoding errors cannot be ascribed to the intrinsic error rate of 2.6×10 −5 of the AmpliTaq Gold DNA polymerase only [46] and therefore originate from the altered parts of the DNA template. The analysis of the deletion content of all molecules with two degraded strands allows distinguishing between two groups of molecules.…”

Characterizing pre-transplant and post-transplant kidney rejection risk by B cell immune repertoire sequencing

“…In all, 0.4 μl of each first PCR product templated the second PCR reaction using external primers specific for the 454 linker sequences; the amplification was carried out with the program: 94 °C for 15 min, 12 cycles of (94 °C for 30 s, 60 °C for 45 s, 72 °C for 90 s), and final extension at 72 °C for 10 min. The error rate of the AmpliTaq Gold should have a minimal effect on the identification of clonally related sequences or estimation of somatic mutation rates in sequences as discussed somewhere else 56 . cDNA was synthesized from total 300 ng of RNA with priming by random hexamers.…”

Single-molecule enzymology based on the principle of the Millikan oil drop experiment

“…The bead suspension was rinsed one time with 15 uL 1M NaCl/5 mM Tris to remove unbound oligonucleotides, then resuspended in 15 uL of 0.1 M NaOH to denature the DNA. After 10 minutes, the NaOH supernatant, which should contain free single stranded PCR product, was removed and 12 uL 0.4 M HCl and 3 uL 1 M Tris, pH 7.0, were added to the NaOH solution for a final pH less than 7.5 [12]. Next, 0.45 uL of 10 uM oligonucleotide A (the suflhydryl-containing capture oligonucleotide, which has the same sequence as the biotinylated primer, A’) was added to the single stranded solution.…”