Restriction Fragment Length Polymorphism Lab Report

Restriction Fragment Length Polymorphism and Southern Blotting

1. Abstract

The aim of the experiment was to be introduced to the techniques involved in the identification of restriction fragment length polymorphisms. Restrictions were carried out using three different restriction enzymes, ECORI, HindIII and BstELI with their buffers. Lambda (λ) DNA was then examined using electrophoresis and Southern blotting.

The results showed that λ DNA was best digested by EcoRI as all of the expected bands can be seen and are in the right place. The HindIII digestion was second best as there was a large amount of merging and the bands contained a lot more streaking compared to the other enzymes. The BstELI was only partially digested. Many bands had not separated, and the other bands were either in the wrong location or barely visible.

2. Introduction

2.1 RFLP

Restriction fragment length polymorphisms (RFLPs), were developed by Botstein et al. in 1980. They are differences in the lengths of DNA fragments cut by restriction enzymes at specific sequences called restriction sites.
After a segment of DNA has been cut, the fragments can be examined using gel electrophoresis. This separates DNA fragments according to their size, forming bands at particular distances along the gel. Longer DNA molecules will not move as far as shorter molecules as the longer ones are heavier and take up more space, therefore they will experience more resistance by the gel. (Poltronieri & Hong, 2015)

Figure 1. Electrophoresis diagram.

If there are differences in the DNA sequences of two individuals at specific restriction sites, their DNA will be cut into fragments of varying lengths by restriction enzymes. Differences can also be observed in the number of DNA fragments among individuals. RFLP analysis can be used detect genetic mutations and diseases to determine whether an individual carries a mutant gene for a disease that runs in their family.

2.2 Southern Blotting

Southern blot was developed by Edward M. Southern in 1975. Southern blotting is used in molecular biology for the detection of a specific DNA sequence in DNA samples. After the fragments have been separated by electrophoresis, they are transferred to a filter membrane, followed by hybridisation with radioactive DNA probe.

Southern blotting can be used to determine the molecular weight of a restriction fragment and compare relative amounts of different samples. Also, it can locate a particular sequence of DNA within a complex mixture and confirm the identity of a cloned fragment.

Figure 2. Southern blotting diagram. (Nickle & Barrette-Ng, 2017)

2.3 Relevance to Forensic Science

RFLP though not widely used now, was one of the first techniques used for DNA analysis in forensic science.
Dr. Alec Jeffreys used RFLP to analyse DNA and discovered that repetitive patterns of DNA were present in all human beings, but they were different in length for all individuals. He realised that the variation could be used to ascertain the identity of a person and called his technique genetic fingerprinting. The process is used as a means of identification when an assailant has left some kind of bodily fluid at the crime scene but no visual identification is possible. Genetic fingerprinting relies on the principle that apart from identical twins no other individuals share the same genetic code. Jeffreys' technique was catapulted into the forensic science domain when two murders were committed and genetic fingerprinting was used to exonerate a suspect and convict the guilty
person.

Another application is paternity. The technique can be used in the determination of paternity or for tracing ancestry. RFLP can also be used for genetic diversity. The evolution and migration of wildlife can be studied as well as breeding patterns in animal populations. It can also be used for the detection and diagnosis of certain diseases.

3. Method

3.1 Preparing a Restriction Digest

Restrictions were carried out using three different restriction enzymes, ECORI, HindIII and BstELI with their buffers.

Table 1. Preparation for the restriction digest

The restriction reaction contained the following:
DNA to be restricted 2 µl
Buffer 1 µl
Restriction Enzyme 1 µl
Water 6 µl
Final volume 10 µl

The restriction reactions were prepared in labelled Eppendorf tubes. A new tip was used for each reagent added. The reagents were added in the order of water, λ DNA, buffer and then restriction enzyme. The tubes were flicked to ensure everything was mixed. They were then centrifuged in order to collect everything in the bottom of the tube.

The tubes were vortexed to make sure they would mix well. The EcoRI and HindIII reactions were incubated for 1 hour in a 37C water bath while the BstELI reaction was incubated for 1 hour in a 60C water bath. Before loading the reaction to the DNA gel, 5 µl of loading dye were added to the tube. This was then mixed and centrifuged, then all 15 µl were loaded onto the gel.