Allele-Specific Loop-Mediated Isothermal Amplification for the Detection of IVSII-I G>A Mutation On β-Globin Gene

Thalassemia is one of the most common genetic health problems in the world. More than 200 different mutations have been identified in the beta-globin gene and among the 24 β-globin gene mutations in β-thalassemia carriers in the north of Iran IVSII-I G>A mutation has the highest frequency. Using fast, inexpensive, simple and reliable methods for the detection of the mutations in β-thal carriers is very important in prenatal diagnosis, and introduction of alternative methods to the existing ones can help to simplify the detection of mutations. Since its introduction, different methods derived from LAMP have been widely used for SNPs detection.

This study was aimed to design a new method for the detection of IVSII-I G>A mutation on β-globin gene based on AS - LAMP technique.

Primer explorer V5 software was used for the design of LAMP primers. Three sets of primers were designed. In the first set, the BIP primers were exactly complementary to the normal and mutant alleles. In the second set, 1 nucleotide (T) was inserted at the 5' end of BIP primers, and in the last set, one nucleotide at the 5' end of BIP primer was changed. The other required primers for the LAMP reaction (FIP, B3, and F3) were the same for all 3 sets of primers. The LAMP reaction was applied on three DNA samples (Wild type, Heterozygote and Homozygote for IVSII-I G>A mutation) and synthetic DNA.

The results of the present study showed that LAMP reaction using three sets of primers could not successfully detect the IVSII-I G > A mutation among subjects DNA sample and synthetic DNA.

Although several studies have successfully used ARMS-LAMP method to detect the SNPs, and other studies use a variety of methods to identify IVSII-I G>A mutation, the present study was unable to differentiate between a normal allele and IVSII-I G>A mutation. Hence further studies are recommended to consider redesigning of primer set, DNA concentration and using commercial LAMP Master Mix to detect the mutation.