MLPA-enhanced specific method for detecting SNP sites

Abstract

The invention discloses an MLPA-enhanced specific method for detecting SNP sites. An MLPA probe aiming at Kras gene 216G-CSNP sites is designed, an MLPA experiment is used, and the influence on the SNP sites during detection is observed. The key points of the method disclosed by the invention are that the mismatch rate of the probe on the SNP sites is reduced when the specificity is detected at the SNP sites, and the probe can be synthesized by using nucleotide subjected to specific chemical modification. The research proves that after the SNP sites of the MLPA probe are modified by using LNA, the base mismatch rate can be obviously reduced by virtue of LNA modification, and the false positive product peak is eliminated; however, the detection sensitivity of the LNA modified probe is reduced, and when a positive sample is detected, compared with a non-modified MLPA probe, the probe disclosed by the invention has the advantages that the product peak area is reduced, probe hybridization or continuous efficiency reduction can be possibly caused by LNA modification, and the detection sensitivity of MLPA is reduced.

Description

technical field [0001] The invention relates to a specific method for enhancing MLPA detection of SNP sites, which belongs to the technical field of gene detection. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) is a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, including substitutions, inversions, deletions, and insertions, and any allele is present in the population. The frequency is not less than 1%. With the completion of the Human Genome Project and the mapping of the SNP database HAP-MAP, SNP detection is playing an increasingly important role in the fields of clinical molecular diagnosis, forensic identification, and new drug development. The common SNP detection Methods include DNA chip hybridization, restriction length polymorphism (restriction fragment length polymorphism, RFLP) and allele-specific PCR (allelespe-cific PCR, AS-PCR), etc., but these methods cannot simultaneo...

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Problems solved by technology

[0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) is a DNA sequence polymorphism caused by a single nucleotide variation at the genome level, including substitutions, inversions, deletions, and insertions, and any allele is present in the population. The frequency is not less than 1%. With the completion of the Human Genome Project and the mapping of the SNP database HAP-MAP, SNP detection is playing an increasingly important role in the fields of clinical molecular diagnosis, forensic identification, and new drug development. The common SNP detection Methods include DNA chip hybridization, restriction length polymorphism (restriction fragment length polymorphism, RFLP) and allele-specific PCR (allelespe-cific PCR, AS-PCR), etc., but these methods cannot simultaneously meet the requirements of high throughput and high accuracy. degree and low cost on request

Multiplex ligation-dependent probe amplification (MLPA) technology was invented by Schouten in 2002, which can detect the copy number changes of more than 40 target sequences at the same time, and the improved MLPA technology can also detect RNA and SNP positions at the same time. It has been widely used in the fields of prenatal, genetic and tumor detection, although the probe ligation reaction in the MLPA reaction can only be performed when the probe and the target DNA are completely matched, and the difference between the two probes However, due to the possibility of mismatching between bases, and the difference of only one base in the SNP sequence, MLPA often produces false positive results due to mismatched connections when detecting SNPs, which affects the interpretation of results.

Such false positive problems caused by base mismatches have been improved in AS-PCR by base modification, adding blocking probes, etc., but have not been resolved in MLPA.

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