Restriction Fragment Length Polymorphism

/Restriction Fragment Length Polymorphism
Restriction Fragment Length Polymorphism 2018-02-24T07:26:18+00:00

Restriction Fragment Length Polymorphism

Digestion of extracted plasmid

Introduction

Restriction fragment length polymorphism (RFLP) method in molecular biology was evolved for detecting variation at the DNA sequence level of various biological samples. The principle of this method is based upon the comparison of restriction enzyme cleavage profiles following the existence of a polymorphism in a DNA sequence related to other sequence. In RFLP, DNA of individuals to be comparedis digested with one or more restriction enzymes and the resulting fragments are separated according to molecular size using gel electrophoresis along with a molecular weight marker. Through this approach two individuals can present different restriction profiles.

Restriction fragment length polymorphism (RFLP) analysis is extensively used in molecular biology for detecting variation at the DNA sequence level. The principle of this analysis is to compare restriction digestion profiles of DNA samples isolated from different individuals. RFLP functions as a molecular marker as it is specific to a single clone/restriction enzyme combination.Most RFLP markers are co-dominant and highly locus-specific. In molecular biology, restriction fragment length polymorphism, or RFLP is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologousDNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated.

RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and digest DNA wherever a specific short sequence occurs, in a process known as restriction digestion. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis. Molecular markers are used to estimate the fragment size. RFLP is specific to a single clone/restriction enzyme combination and it occurs when the length of a detected fragment varies between individuals.

Requirements

Reagents

  1. Agarose
  2. DNA Staining Solution
  3. Electrolysis Buffer
  4. 6x Gel-Loading Buffer
  5. Test DNA Sample
  6. Reference samples
  7. DNA Ladder
  8. 10x Assay Buffer
  9. Milli Q Water
  10. EcoRI
  11. Pstl
  12. Horizontal electrophoresis buffer
  13. Power supply

Procedure

  1. Before starting the experiment, crush ice and place the vials containing DNA samples, restriction enzymes and assay buffers onto it.
  2. In this experiment three reference DNA samples and the test sample are digested simultaneously with two restriction enzymes EcoRI and PstI.
  3. Set up four separate reaction mixtures as follows:
  • DNA sample: 15.0 μl
  • 10X Assay Buffer: 3.0 μl
  • Milli Q water: 10μl
  • EcoRI: 1.0μl
  • PstI: 1.0μl
    Total: 30μl
  1. After preparing the four reaction tubes, mix the components by gentle pipetting and tapping.
  2. Incubate the tubes at 37oC for 2-3 hours.
  3. Meanwhile, prepare 1.5% agarose gel for electrophoresis.
  4. Add 5 μl of Gel Loading Buffer to each tube.
  5. Load 5 μl of DNA Marker into the well 1. Load 30 μ l of each DNA samples (reference sampl es) onto wells 2, 3 and 4. Load 30 μl of test DNA sample well
  6. Electrophorese the samples at 100-120 V for 1-2 hours.
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