Open AccessMismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens
Author(s) -
Vijaya Kumar Deekshit,
Kadeeja Jazeela,
Gunimala Chakraborty,
Anusha Rohit,
Anirban Chakraborty,
Indrani Karunasagar
Publication year - 2019
Publication title -
indian journal of medical research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.578
H-Index - 87
ISSN - 0971-5916
DOI - 10.4103/ijmr.ijmr_2091_17
Subject(s) - polymerase chain reaction , point mutation , multiple displacement amplification , primer (cosmetics) , biology , hot start pcr , primer dimer , oligonucleotide , microbiology and biotechnology , in silico pcr , restriction fragment length polymorphism , polymerase , genetics , taq polymerase , restriction site , multiplex ligation dependent probe amplification , primer extension , multiplex polymerase chain reaction , gene , mutation , restriction enzyme , nucleotide , chemistry , dna extraction , thermus aquaticus , organic chemistry , exon
The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.