Federal University of Pelotas Oncology Research Group, Technology Development Centre (Biotechnology Unit), Federal University of Pelotas, Pelotas, Brazil
* Corresponding author Email: firstname.lastname@example.org
Genetic factors play important roles in cancer and other complex diseases. An important aspect of genomics is genotypic phasing, which has important functional implications. Although there are recently developed methods for whole genome haplotyping, it is still of interest to consider targeted haplotyping approaches in some situations. Methodologies such as allele-specific polymerase chain reaction and single molecule dilution have been proposed for this purpose more than 20 years ago. However, each of these methodologies presents particular difficulties or limitations. In this study, an alternative method for allele-specific amplification for targeted haplotyping is proposed.
The method was presented in a hypothetical situation in the clinical setting, and can be divided into three steps: (1) Genotype all the markers (or sequence an entire target region); (2) If phase ambiguity is observed, amplify the entire region in a reaction combining two key factors: a non-extendable oligonucleotide probe that binds only to one of the alleles of one of the heterozygous markers, and a 5′→3′ exonuclease activity-lacking
The presented method, although a hypothesis (has not been formally tested for targeted haplotyping yet), is based on well-established concepts of polymerase chain reaction and probe-based single nucleotide polymorphism genotyping. It has the potential to overcome both allele-specific polymerase chain reaction (it has a highly discriminatory capacity, does not require labourious and time-consuming standardisation and can make use of genotyping assays validated for a huge number of single nucleotide polymorphisms in the genome) and single molecule dilution (it is technically more straightforward, less time-consuming and requires a simpler infrastructure), indicating its adequacy for haplotyping applications in the clinical setting.
Although there are other methods already proposed for allele-specific polymerase chain reaction for targeted haplotyping applications, the proposed probe-based method (which can be combined with sequencing for detecting or