Saturday, June 22, 2013

Application of Restriction Fragment Length Polymorphism (RFLP)

DNA-based screening is useful not only in determining if an unborn fetus is affected, but also in detecting carriers of the mutated gene. PKU, like many inborn errors of amino acid metabolism, is inherited as an autosomal recessive trait. Identification of heterozygotes can aid in future family planning.

Another approach of detecting the βs globin mutation is by the use of Allele specific oligonucleotide probe (ASO). If PCR is carried out using such primer then it is called ASO-PCR.

DNA, isolated from white blood cells, is denatured into single strands. An oligonucleotide is constructed that is complementary to the portion of the mutant globin gene coding for the amino-terminal sequence of the β-globin protein. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell disease) contains a nucleotide sequence that is complementary to the probe. Thus, a double-stranded hybrid forms that can be detected by electrophoresis.

Fig: Analysis of restriction fragment length polymorphism in a family with a child affected by phenylketonuria. The molecular defect in the phenylalanine hydroxylase (PAH) gene in the family is not known. The family wanted to know if the current pregnancy would be affected by phenylketonuria.
In contrast, DNA obtained from normal individuals is not complementary at the sixth nucleotide triplet (coding for glutamate in normal individuals but for valine in patients with the βS gene) and, therefore, does not form a hybrid . Use of a pair of such ASO (one specific for the normal allele and one specific for the mutant allele) allows one to distinguish the DNA from all three possible genotypes—homozygous normal, heterozygous, and homozygous mutant.

Restriction Fragment Length Polymorphism (RFLP) and Single Nucleotide Polymorphisms (SNPs):

Variation in DNA sequences can result in variations of restriction sites and thus length of restriction fragments. Similarly SNPs can be detected by PCR. An inherent difference in the pattern of restriction enzyme digestion (e.g. There is 0.1% DNA sequence variation occurring in genome of non related people) between individual is known as restriction fragment length polymorphism or RFLP. RFLPs results from single base changes (e.g. in sickle cell disease) or from deletions or insertions (CNVs) of DNA into a restriction fragment (eg, the thalassemias) or due to polymorphism, that will create or abolish the restriction site in the genome. Polymorphism are found in known gene loci and in sequences that have no known functions; thus RFLPs may disrupt the function of the gene or may remain silent.

DNA Variation resulting in RFLP:

Two types of DNA variation commonly result in RFLPs: single-base changes in the nucleotide sequence, and tandem repeats of DNA sequences.

Single base changes in DNA: About 90% of human genome variation occurs in the form of single-nucleotide polymorphisms, (SNPs, pronounced “snips”), that is, variations that involve just one base. The alteration of one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths differing from the normal, which can be detected by  DNA hybridization like southern blotting technique. [Note: The altered restriction site can be either at the site of a disease-causing mutation or at a site some distance from the mutation.]

Tandem repeats: 
Alternatively, polymorphism in chromosomal DNA can arise from the presence of a variable number of tandem repeats (VNTR). These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person, but is unique for any given individual and, therefore, serves as a molecular fingerprint. Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment. Variation in the number of tandem repeats can lead to polymorphisms. Many different VNTR loci have been identified, and are extremely useful for DNA fingerprint analysis, such as in forensic and paternity identity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers and do not code for any proteins.

Microsatellite DNA Polymorphisms: Microsatellite DNA are Short (2-6 bp), inherited, tandem repeat units of DNA occur about 50, 000 – 100, 000 times in human genome. Since they occur frequently they are replacing RFLPs as the marker of loci for various genome searches.

RFLPs and SNPs are inherited and segregate in Mendelian fashion. They are used in inherited disease in which functional deficit is unknown. These can be used to establish linkage groups, which in turn, by process of chromosome walking will define the disease locus. In chromosome walking a fragment of DNA out of large piece of DNA is used to isolate another that overlaps but extends the first.

E.g. X chromosome-linked disorders are identified by chromosome walking, since only a single allele is expressed. Hence 20% of the defined RFLPs are on the X chromosome. The gene for X- linked disorder, Duchenne-type muscular dystrophy was found using RFLPs. Defect that cause polycystic kidney disease is linked to α-globin locus on chromosome 16.

The technique of chromosome walking. Gene X is to be isolated from a large piece of DNA. The exact location of this gene is not known, but a probe (*——) directed against a fragment of DNA (shown at the 5′ end in this representation) is available. The initial probe will hybridize only with clones containing fragment 1, which can then be isolated and used as a probe to detect fragment 2. This procedure is repeated until fragment 4 hybridizes with fragment 5, which contains the entire sequence of gene X.

(Source: Lehninger's Textbook of Biochemistry, Lippincott's Illustrated Biochemistry and Practical Biochemistry)
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