Mutation detection methods

Mutation in genes are responsible for several genetic disorders. Identifying these mutations requires novel detection methods. Intensive research over the years has led to several new detection techniques. Some of them which are specific for breast cancer are discussed in this section. These tests are done taking into account, the patient's cancer history and family history .

The basic principles involved in these techniques are discussed here.

Allele Specific Oligonucleotides:

This method is used to detect one or more known specific mutations in population. It makes use of nucleotide probes which are short and specific for particular DNA sequences. This is a simpler and less expensive approach than searching for any new mutation in family members. This type of mutation detection technique is more popular in Ashkenazi jewish population and other founder mutations.
The major disadvantage of this technique is that it uses radio active material and can miss other mutations if present. [in detail]

Protein truncation test (PTT):

Most BRCA1 and BRCA2 mutations result in short or truncated protein. PTT is a method that detects mutations arising from termination of mRNA translation process. As a result of this, the protein product is truncated. The truncated protein could have arised due to a frameshift mutation, non-sense mutation and splice site mutation. The advantage of PTT method is that, it can detect mutations of large kilobases. However, this method cannot detect polymorphisms, silent mutations and missense mutations. [in detail]

Single Strand Conformational Polymorphism:

This method can detect single base pair mutations like frameshift mutations, nonsense mutations and missense mutations in breast cancer. The basic principle of this test is that double stranded DNA when denatured, assumes a special conformation. This conformation is unique and depends on primary nucleotide sequence. This method is quite sensitive to detect even single nucleotide difference that occupies a different conformation and when subjected to electrophoresis, the variant nucleotide occupies a different position. [pubmed]

Nucleotide sequencing:

In this method, the complete gene is sequenced to identify the presence of any mutation in breast cancer. Till date complete gene sequencing is considered as a gold standard method because any type of mutation can be identified. Sequencing is done by two methods:
  • Sanger's dideoxy nucleotide method
  • Shotgun method

Denaturing Gradient Gel Electrophoresis (DGGE):

DNA is first extracted either from paraffin embedded or fresh tissues and subjected to denaturing gradient gel electrophoresis. The method detects selective retention of mutated BRCA1 alleles, based on the principle that different sequences of double-stranded DNA melt under different conditions. A modified form of DGGE is called as TGGE (Temperature Gradient Gel Electrophoresis) which uses temperature as a denaturing agent. [clinical chemisry]

Heteroduplex analysis (HDA):

The principle behind this technique is that, DNA strands can be separated from each other upon denaturation. Similarly, it can also be renatured, so that the DNA strands come together, in other words, reassociate themselves to form a homoduplex. But if there is a mutation in one of the strands, then the resultant DNA is in the heteroduplex state. Such property of DNA can be best exploited for detecting point mutations. Furthermore, the heteroduplex DNA strands move slower on a polyacrylamide gel when compared to homoduplex DNA. [pubmed]

DNA microarray technology:

Base-pairing or complementarity is the principle behind this emerging technology. The potential applications of microarray technology are gene expression profiling and identification of gene sequences (including sequences that bear mutations). In this method, a large number of DNA fragments are placed on a glass slide. The fragments are allowed to complement or bind with the labeled DNA (probes), which hybridize with the DNA on the glass slide. The amount of hybridization is then analyzed in each spot on the slide. The genes are given a color where the hybridized ones are coloured red and the genes that are hybridised least are colored green. A detailed account of this latest technology is given in this webpage. It has been reported that microarray technlogy could be used in monitoring chromosome gains and losses, tumour classification, drug discovery and development. Also the technique comes handy in detecting mutations and further investigating the mechanism of tumour development. [pubmed]