Point Mutation Studies: Methods of detection

Introduction

Mutations are any possible alterations in DNA sequence and sequence organization, from point mutations to genome structural variation, chromosomal aberrations, and aneuploidy. Epi-mutations are defined as alterations in the epigenome, i.e., changes in DNA methylation, histone modification, and small regulatory RNAs. We are fascinated by mechanisms of epi-mutation induction, for example, during DNA repair, replication, or recombination; the landscape of somatic mutations and epi-mutations in cancer and aging; the role of de novo mutations in human disease and aging; the role of epi-mutations in chromatin structure and function; mitochondrial DNA mutations and their consequences in terms of human disease and aging; and novel ways to generate mutations and epi-mutations in cell lines and animal models. The study of genome instability in human molecular epidemiology and in relation to complex phenotypes, such as human disease, is considered a growing area of importance. There are different methods for the detection of point mutations and small deletions or insertions which are listed below.

Methods

In general, PCR is either used for the generation of DNA fragments or is part of the detection method. Mutations assumed from the results of screening methods must be confirmed by DNA sequencing. Disregarding direct sequencing of PCR products, two different approaches for the detection of unknown point mutations can be distinguished. One set of methods relies on the differences in electrophoretic mobilities of wild-type and mutant nucleic acids. The second group of methods is based on the cleavage of heteroduplexes. Recently, a new principle that depends on the association of mismatch binding proteins with mismatches in heteroduplexes has been described.

In general, target sequences are amplified by PCR before analysis. At present, Taq polymerase is widely used for amplification. The error rate of Taq polymerase is in the range of 10−4 to 10−5 per nucleotide and is strongly affected by the reaction conditions (e.g., concentrations of magnesium chloride and dNTPs, pH, and temperature). Depending on the method of choice, polymerase errors may contribute reasonably to the unspecific background, limiting the level of detection and thermostable polymerases with higher fidelity (e.g., Pfu DNA polymerase) may improve results in particular applications.



1) Denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) - With increasing concentration of denaturant or temperature, domains in the DNA dissociate according to their melting temperature (Tm). DNA hybrids of 100–1000 bp contain 2–5 such domains, each melting at a distinct temperature. Dissociation of strands in such domains results in a decrease in electrophoretic mobility. The maximum fragment size suited for DGGE is ∼1000 bp. With an increasing number of melting domains, the mobility shifts decrease.

2) Single-Strand Conformation Polymorphism (SSCP) - Under certain conditions, single-stranded nucleic acids form secondary structures in solution. The secondary structure depends on the base composition and may be altered by a single nucleotide exchange, causing differences in electrophoretic mobility under non-denaturing conditions. For optimal results, fragment size should be in the range of 150 to 200 bp.

3) Heteroduplex Analysis (HET) - The optimal fragment length for the detection of point mutations varies between 200 and 600 bp; the detection of mutations in PCR fragments of up to 900 bp has been reported.

4) RNase A cleavage method - Under defined conditions, mismatches within RNA-RNA or RNA-DNA heteroduplex are cleaved by RNase A. After cleavage, labeled fragments are analyzed by denaturing gel electrophoresis. The maximum size of RNA that can be analyzed is ∼1000 bp.

5) Chemical Cleavage Method (CCM) - In principle, all possible mutations are detectable by CCM

6) Enzyme Mismatch Cleavage (EMC) - Mutations were detectable in PCR products between 88 and 940 bp or up to 1.5 kb.

7) Cleavage fragment length polymorphism (CFLP) - Fragments of up to 2 kb can be analyzed.

8) Mutation detection by mismatch binding proteins

9) Allele-specific oligonucleotide (ASO) hybridization on DNA chips

10) Protein Truncation Test (PTT)

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