Rlp Analysis Of Dna Fingerprinting

Diarmuid Scanlon

13333181

RFLP Analysis and VNTRs

Introduction

DNA fingerprinting is a technique used to identify individuals based on their specific DNA profile. This technique was first discovered in 1986 by Sir Alec Jeffreys, a British geneticist at the University of Leicester. He was interested in solving immigration and paternity disputes by confirming the genetic links between individuals. Jeffreys analysed DNA using a method called Restriction Fragment Length Polymorphism (RFLP). RFLP analysis was the first method in DNA fingerprinting to be used widely due to its cost effectiveness.

Sir Alec Jeffreys - The Pioneer of DNA Fingerprinting

During his research, Jeffreys observed that repetitive patterns of DNA called Variable Number

For this reason the DNA is denatured into single strands by incubation with sodium hydroxide solution (NaOH). If the DNA fragments are larger than 15kb, the gel can be treated with acid such as dilute HCl prior to blotting. The DNA is depurinated by the acid and breaks down into smaller pieces which allows more efficient transfer from the gel to the membrane.

The Southern Blot Method begins with the DNA fragments being transferred to a nitrocellulose membrane. This is a sheet of special blotting paper. The DNA fragments keep the same pattern of separation that they had on the gel. The blot is incubated with multiple copies of a probe. The probe forms base pairs with the complementary the DNA sequence and binds to form a double stranded DNA molecule. This step is called hybridisation.

After hybridisation, SSC buffer is used to wash excess probe from the membrane. Autoradiography is used to visualise the hybridisation pattern. When the probe is incubated with a colourless substrate, its location is revealed. The attached enzyme converts the substrate to a coloured product that can be seen or releases light when exposed on x-ray film. The probe can be exposed on x-ray film directly if it's labelled with

The VNTRs alleles from both samples must share the same specific location.

• Inheritance matching: here, the rules of inheritance must be followed by the VNTRs. In a paternity test for example, the child must have an allele that matches one from each parent. When relationships are more distant e.g. sibling, then matches must be constant with the degree of relatedness.

Disadvantages of RFLP Analysis

RFLP analysis is useful for mapping the human genome and comparing DNA but this technique is less widely used these days. It is a slow and tedious process as the Southern Blot Method requires a lot of work. The duration of process can take up to 1 month.

It also requires a significantly larger sample size compared to other forms of DNA profiling. The sample size for RFLP would generally have to be the size of a one euro coin. This might sound small but is very large in comparison with other processes such as PCR analysis which only needs a few cells for its sample to be sequenced successfully. Moreover, in RFLP analysis many minisatellite loci are being examined simultaneously which makes it difficult to distinguish individual