Two-temperature LATE-PCR endpoint genotyping

BACKGROUND: In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. RESULTS: Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles. CONCLUSION: SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions

Decreased Mitochondrial DNA Mutagenesis in Human Colorectal Cancer

Genome instability is regarded as a hallmark of cancer. Human tumors frequently carry clonally expanded mutations in their mitochondrial DNA (mtDNA), some of which may drive cancer progression and metastasis. The high prevalence of clonal mutations in tumor mtDNA has commonly led to the assumption that the mitochondrial genome in cancer is genetically unstable, yet this hypothesis has not been experimentally tested. In this study, we directly measured the frequency of non-clonal (random) de novo single base substitutions in the mtDNA of human colorectal cancers. Remarkably, tumor tissue exhibited a decreased prevalence of these mutations relative to adjacent non-tumor tissue. The difference in mutation burden was attributable to a reduction in C∶G to T∶A transitions, which are associated with oxidative damage. We demonstrate that the lower random mutation frequency in tumor tissue was also coupled with a shift in glucose metabolism from oxidative phosphorylation to anaerobic glycolysis, as compared to non-neoplastic colon. Together these findings raise the intriguing possibility that fidelity of mitochondrial genome is, in fact, increased in cancer as a result of a decrease in reactive oxygen species-mediated mtDNA damage

Clonal evolution in cancer

Cancer is a disease of somatic evolution. Random mutations that arise during life and confer a growth advantage upon a cell will preferentially multiply to form a tumor. This dissertation considers some of the implications of clonal evolution in cancer with an emphasis on how the evolutionary process, itself, facilitates disease progression and how its signature can be clinically used for early disease detection. The first part introduces the subject of mutations in cancer within an evolutionary context and discusses the origins and consequences of genetic heterogeneity in developing neoplasms. The second part describes experimental studies on clonal evolution of two distinct varieties. In the first study, somatic mutations in mutational hotspots are used to detect preneoplastic clonal expansions in the colons of patients with the cancer-predisposing disease, ulcerative colitis (UC). The results show that clones within non-dysplastic UC tissues are strongly correlated with cancer progression elsewhere in the colon and are potentially a biomarker of future cancer development risk. The second study describes competition experiments among E. coli strains with differing mutations rates as a model of competition among genetically unstable tumor cells. The results show that an optimal mutation rate exists for cells competing under specific conditions and that deviation in either direction from this optimum is detrimental to evolvability. The findings suggest that targeting mutation rate may be one means by which the evolutionary process of cancer progression can be delayed. Together this work contributes to the growing body of knowledge of the significance of clonal evolution in cancer and lays the groundwork for future studies to translate these findings into clinical tools and treatments

Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage

<div><p>Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10⁷ wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ∼5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G→T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G→A and T→C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.</p></div

Overview of the Duplex Sequencing methodology.

<p>(A) Adapter design with random double-stranded tag sequence and invariant spacer sequence. (B) Ligation of adapters to fragmented DNA generates unique 12 bp tags on each end (α and β). PCR amplification of the two strands produces two related, but distinct products. (C) Sequence reads sharing unique α and β tags are grouped into families of α-β or β-α orientation. Mutations are of three different types: sequencing mistakes or late arising PCR errors (blue or purple spots); first round PCR errors (brown spots); true mutations (green spots). Comparing SSCSs from the paired families generates a DCS, which eliminates all but true mutations.</p

Mitochondrial point mutations increase with age and are biased to transitions.

<p>(A) mtDNA point mutations burden is higher in older individuals (<i>purple</i>) than young individuals (<i>yellow</i>) (p<10⁻⁴, two-tailed t-test). Error bars represent the 95% confidence interval for each sample (Wilson Score interval) (B) The mutation spectra of both young (<i>yellow</i>) and aged (<i>purple</i>) individuals shows an excess of transitions, relative to transversions. Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced. Indels were calculated independently as events per total number of bases sequenced. Error bars represent one standard deviation. (C) The mutation spectra, reported as the relative proportion of the different mutation types, do not change with age. Error bars represent one standard deviation. Significance was tested using the two-tailed t-test.</p

The D-loop has an elevated mutation burden but its mutation spectrum is similar to the remainder of the mitochondrial genome.

<p>(A) The D-loop (<i>orange</i>) exhibits a higher aggregate mutation burden than the rest of the genome (<i>grey</i>). Frequencies were calculated by dividing the number of mutations of each type by the number of times the wild-type base of each mutation type was sequenced. Indels were calculated independently as events per total number of bases sequenced. Error bars represent one standard deviation. (B) The relative fraction of mutations exhibits no difference between the D-loop (<i>orange</i>) and non-D-loop (<i>grey</i>) portions of the genome, suggesting a similar underlying mutagenic process. Error bars represent one standard deviation.</p