Identifying known mutations in single genes

The following PCR-based techniques are used in laboratories to investigate the common mutations.

• ARMS-PCR (Amplification-Refractory Mutation System PCR)
  A terrible name but a simple concept! This PCR technique is a method for detecting mutations that involve a single base change or small deletion. It uses primers that are specific for certain mutations, only resulting in amplification of the PCR product if the mutation is present. It can be used to test for a single mutation, or panel of common mutations (eg in CF testing).
• OLA (Oligonucleotide ligation assay)
  OLA can also be used to test for a group of common mutations. However in OLA it is not the presence or absence of a PCR product that is diagnostic, but the size of the product. PCR primers are used that are complementary to normal or mutant sequences, and these are each labelled with a different-sized tail. One mutation site is therefore defined by two probes. Following hybridization of the PCR primer to the complementary normal or mutant sequence there is a PCR amplification step. Then a second probe is introduced containing a 3’ fluorescent dye (FAM, TET or HEX). Ligation, using a thermostable ligase, is performed between the two probes - this will only occur when the 5’ probe matches perfectly with the target sequence. If there is a mismatch, ligation will not occur (Figure 1). The resultant ligated fluorescent probes are size-separated by electrophoresis and the presence or absence of normal and/or mutant alleles determined (Figure 2).

 Figure 1: In this example, the test DNA does not carry the mutation. There is therefore binding (and subsequent ligation) of the normal primer, but not the mutant primer. 
© St George’s, University of London

 Figure 2: This image shows the result of OLA testing for the common cystic fibrosis (CF) mutations. There is a peak for each normal allele (labeled in black), and an extra peak is seen for each mutant allele (labeled in orange). Here you can see that the patient has two extra peaks, and is a compound heterozygote for the ΔF508 and 621+1G>T mutations.
© St George’s, University of London

• PCR and restriction digest assay

PCR can also be used in conjunction with a restriction digest assay to detect common mutations, and is particularly useful for those that fall outside of normal Sanger sequencing boundaries, for example splice-site mutations.

A good example of the use of PCR and a restriction digest assay is in mutation analysis of the GJB2 gene (also known as connexin 26), a common cause of nonsyndromic sensorineural hearing loss.

Although many GJB2 mutations can be detected by GJB2 sequencing analysis, there is a known pathogenic splice site mutation (c.-23+1G>A) which is not usually included in the analysed region. However, a simple PCR and restriction digest assay can be run alongside the sequencing to detect this mutation.

A fragment spanning exon 1 and the flanking splice site is amplified by PCR. The PCR product is then digested with the HphI restriction enzyme, and the resulting fragments are size separated by gel electrophoresis. Presence of the splice site mutation alters the HphI restriction site, resulting in a larger restriction digestion product for the mutant allele than for the normal allele.

Source:-https://www.futurelearn.com/courses/molecular-techniques/6/steps/765684