Allele Specifc Oligonucleotide Hybridization #Biotech #Medtech #Blog #DNA #Allele

HISTORY:

Synthetic oligonucleotides usage as specific probes for genetic sequence variations was introduced by R. Bruce Wallace who was working at the City of Hope National Medical Center in Duarte, California. Wallace and his coworkers reported in 1979, the use of ASO probes to detect variations in a single-stranded bacterial virus, and later applied the technique to cloned human genes. In 1983 and 1985 Wallace's lab reported the presence of the mutation for sickle cell anemia in samples containing whole genomic DNA, although this application was restricted by the small amount of label that could be carried by the help of ASO.

Pairing of PCR with ASO solved the problem of ASO labeling, as the amount of target DNA could be amplified exponentially. Also, the specificity of the PCR process could be added to that of the ASO probes, greatly minimizes the problem of binding of the ASO to non-target sequences.

BACKGROUND:

ASO is a short oligonucleotides usually ranging between 15-21 nucleotide. It is complementary to either normal sequence or known mutated sequence. ASO is designed for a single allele to detect the presence of a particular mutation. It acts as a probe to detect the presence of certain sequence. ASO hybridization technique is used in genetics, molecular biology, forensics, research.

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METHODOLOGY:

General:

Typically, multiple ASO are required for mutational screening. For adult patients and prenatal diagnosis two probes are required for mutational screening. One for normal DNA and one for mutated one. Genotype of patient is determined by signal received due to ASO binding.

PCR:

Polymerase chain reaction can be used to amplify very minute amount of DNA. Even if a single copy of gene or even part of gene is present, PCR can amplify it to millions of copies. On the other hand, reverse transcriptase PCR can be used to convert RNA to DNA and then is amplified further.

Southern blot assay:

After DNA amplification, ASO can be used to detect the presence of certain target sequence by southern blot assay. ASO being single stranded binds to the complementary sequence. In this case, sample DNA is separated on the basis of size via gel electrophoresis.

Dot blot assay:

ASO are also used in dot blot assay in order to determine particular sequence of interest. In this method, instead of separating DNA on basis of size, whole DNA is pooled on a membrane like nitrocellulose. ASO must be labeled to detect it in its bound form. Size of dot on the membrane tells about the variation among samples.

Reverse dot blot assay:

This technique detects various mutations in single time. In this method various unlabeled ASO are blotted on the membrane. DNA amplified through PCR is then labeled and transferred to nylon membrane for hybridization with complementary ASO.

DNA microarray:

This technique involves blotting of series of several DNA oligonucleotides spots. ASO is used as probe that hybridizes to the complementary sequence and makes double strands which is then detected by attachment of fluorescent dye.

RESEARCH:

ASO techniques have been automated for quick screening of several genetic mutations such as sickle cell anemia. An international scientific project i.e. human genome project has discovered 20 to 25000 genes in human DNA. This increases the list of target DNA.

SHORTCOMINGS:

  • This method does not allow screening populations which carry a large number of different gene alleles. This is because each mutation requires hybridization and washing step. Nevertheless, increased automation of the ASO assay process is giving more rapid results.
  • ASO screening is able to detect only known gene mutations. Other "silent" DNA mutations may complicate ASO screening.
  • The assay is not available for common people use due to association of high costs.
Reginald Swift