Restriction Fragment Length Polymorphism

Molecular biology of the cell with Lab

Instructor: Assem Duisembekova

Section #1

Title: Restriction Fragment Length Polymorphism (RFLP)  

Name: Alibek Ysmaiyl

Introduction

     Restriction fragment length polymorphism, RFLP, is a technique which was invented by English scientist Alec Jeffreys in 1954 when hereditary diseases was under the research of this scientist [1]. RFLP refers to genetic analysis by molecular method in which individuals are determined by unique characters of restriction enzyme cutting only specific DNA regions [1]. As it was mentioned before, RFLP is based on the restriction endonucleases, enzymes that cut given DNA into short pieces, called DNA fragments. Each individual restriction endonuclease targets different nucleotide sequences which allows cutting at different sites [2]. Therefore, the length of produced DNA fragments differs across both individual organisms and species.

     Basically, RFLP consists of a series of steps such as DNA fragmentation, gel electrophoresis, and visualization of bands [2]. First of all, DNA fragmentation is based on the digestion of DNA strand using certain restriction endonucleases. The sites of recognition for these enzymes differ between 4-6 base pairs in length. Hence, shorter the sequence is recognized, greater the number of fragments are generated from digestion by restriction endonuclease [1]. Secondly, produced DNA fragments by restriction endonucleases are analyzed using gel electrophoresis. As it is known, the DNA fragments are negatively charged, and when the electric field is applied, these DNA fragments migrate toward the positively charged electrodes. Smaller fragments move faster through the gel, thus making separate DNA samples on the gel by different bands [1]. Finally, the gel treated with luminescent dye is visualized under the UV light [1].

     There are several applications of RFLP method [3]:

• To show the status of genetic diseases such as Cystic fibrosis in an individual.
• To determine the source of DNA sample in criminal investigations or paternity tests.
• Identification of recombination rates in genetic mapping
• Determination of a disease carrier in a family.

     In spite of its applications, RFLP analysis is still slow and time consuming. Moreover, it requires considerably large sample sizes than other types of analysis. Also, RFLP procedure requires many steps that takes weeks to yield results, whereas methods such as PCR can amplify the DNA sequence in a few hours. The main reason of low usage of RFLP method is replacement of RFLP by the identification of single-nucleotide polymorphisms in Human Genome Project in disease analysis [3].

Materials and procedure

Materials: 1.2% agarose gel, DpnI, 5x loading dye, 10x buffer, dH2O, construct/vector, ice box, pipets, 1 kB DNA ladder, centrifuge, micro centrifuge tubes.

Procedure:

1. Virtual digestion of vector and construct by DpnI was obtained during in class instructions in computer lab session.

2. Next lab session, 1.2% agarose gel was prepared by TA’s.

3. For group “Gamma”, two digestion reactions was performed on vector and construct by DpnI restriction enzyme.

4. Namely, into micro centrifuge tube labelled as “V”, 12 ul of dH2O, 2 ul of 10xbuffer, and 5 ul of vector was added. After that, 1 ul of DpnI restriction enzyme was added into tube in ice box. The same procedure was performed for construct by adding 5 ul of construct instead of 5 ul of vector.

5. Then, controls were prepared for vector and construct, by adding 13 ul of dH2O, 5 ul of construct/vector, and 2 ul of 10xbuffer into micro centrifuge tubes for vector control and construct control respectively.

6. All controls and digested tubes were spin down in centrifuge, and incubated at 37oC for 30 minutes.

7. 4 ul of 5x loading dye was added to each tube, and mixed. Finally, 20 ul samples from each tube were added onto the gel. 1 kB DNA ladder was also added to the first wall, and then controls were added to 2 and 3 walls respectively.

8. Gel was run at 100V for 40 minutes, and gel was photographed.

9. Virtual digest and real gel was compared according to their fragmentation and size.

Results

Table 1.Restriction table for DpnI enzyme used for vector

[pic 1]

This restriction table shows the number of cuts performed by DpnI on vector, and cut nucleotide sequence is shown by start and end of cut.

Table 2.Restriction table for DpnI enzyme used for construct

[pic 2]

This restriction table shows the number of cuts performed by DpnI on construct, and cut nucleotide sequence is shown by start and end of cut.