Restriction Fragment Length Polymorphisms (RFLP) and PRC . techniques

RFLP is defined as restriction fragment length polymorphism, representing the difference in size of segments produced when DNA is cut by restriction enzymes when there is a change in the sequence of genomic DNA. nucleus or in other organelles.
RFLP is the most commonly used technique today. The principle of this technique is based on the specificity of restriction enzymes (REs) with respect to their recognition sites on genomic DNA. Genomic DNA will be cut with restriction enzymes, run through an agarose gel electrophoresis, permeable through a hybrid membrane, and hybridized with a (radiolabeled) DNA probe bound to a particular locus. The difference in the cutting position between the two individuals will result in different cutting segments.

RFLP has the advantage of being a co-dominant marker that allows homozygous and heterozygous individuals to be distinguished. Due to the large size of DNA investigated in RFLP, the number of markers generated is large enough to meet the research needs. However, due to the complicated procedure, dangerous to the health of researchers (using radioactive markers), high-quality DNA is required, which limits the use of this technique.
With the development of PCR techniques, RFLP techniques became simpler. An oligonucleotide primer pair can be used to amplify a region of DNA to be examined, then the amplified DNA is cut with REs, electrophoresis and analyzed on ethidium bromide or silver staining gels. PCR-RFLP skips the hybridization step, so it is cheaper and less dangerous than the RFLP method.

RFLP technique is a technique to study the length polymorphism of DNA segments based on the cut-off point of restriction enzymes (Restriction Enzyme, RE). When incubating DNA with restriction enzyme in the appropriate buffer solution at pH, the right temperature will produce DNA fragments with different sizes, thereby creating gene maps. This technique has been widely used since the 80s until now.

II. General principles

The principle of this technique is based on the specificity of restriction enzymes (REs) for their recognition sites on genomic DNA. The difference in the cutting position between the two individuals will result in different cutting segments.

III. Restriction Enzymes (REs)

1. Introduction

Restriction enzymes were found in bacteria by Werner Arber in 1962. He suggested that REs have a very special ability that is the ability to recognize host DNA and foreign DNA. This enzyme restricts the replication of foreign DNA when they enter bacterial cells by specifically cutting them into fragments, so he called restriction meaning restriction. For this invention he and his collaborators (Daniel Nathans and Hamilton O. Smith) won the Nobel Prize in 1978.

REs constitute a defense system in procaryotic cells, one question is why REs only cut the DNA of phages but not the DNA of bacterial cells? The answer is that thanks to Methylase, this is an enzyme that helps bacteria to have this ability, it is this enzyme responsible for attaching the methyl group (CH3) to the nucleotides at the cut site of the REs on the bacterial DNA, thus protecting the DNA of the bacteria. bacteria from cleavage of RE. However, sometimes the methylase binds the methyl group to the phage’s own DNA, which explains why the cells that are resistant to phage are still degraded.

The first restriction enzyme was isolated in 1968, about 3,400 REs have been discovered so far. Of these, about 540 RE have been commercialized.

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