Restriction Fragment Length Polymorphism

...

Figure 1.Virtual digest for digestion of vector and construct

[pic 3]

This virtual digest shows the size of DNA fragments produced by DpnI restriction enzyme on vector and construct. It can be noticed that DpnI made more cuts on construct than on vector.

Figure 2.Gel digestion for vector and construct

[pic 4]

In this gel, 1 kb ladder is clearly seen on the right side, and uncut vector and construct are shown as controls. Digested vector with DpnI is clearly shown around 789-934 bp, however, digested construct did not show any bands on the gel, and will be explained in discussion part

Discussion

According to table 1 and 2, the restriction tables for DpnI enzyme on vector and construct are shown respectively. So, DpnI enzyme made 23 cuts on vector and 39 cuts on construct. Also, the ideal conditions for these cuts are shown in these tables. Basically, DpnI enzyme cuts between adenine (A) and thymine (T) nucleobases.

In figure 1, DNA fragmentations of vector and construct by DpnI enzyme are shown. So, under ideal conditions there would be 10 fragments from vector digestion and 18 fragments from construct by DpnI restriction enzyme. Also, in virtual digest of construct there are some single thick bands which indicate that two or more bands co-localizing.

In figure 2, the real DNA fragmentation is shown, and according to results digested vector shows almost the same sizes of DNA fragments as it is on virtual digest, and 3 bands are demonstrated in both virtual and real gels around 789-934 bp. However, less than 789 bp DNA fragments are not shown in real gel, whereas all DNA fragments from digested vector are shown in virtual digest. This can be explained by the fact that bands smaller than 500 bp might have run off the end of the gel and therefore no longer present in real gel in comparison to virtual digest. In addition, virtual digest shows 18 fragments for digested construct, whereas real gel no longer shows any DNA fragments. The main reason might be the usage of 6x loading dye. So, 5x loading dye were used for controls and digested vector. Unfortunately, 5x loading dye have run off, and it was suggested to use 6x loading dye for digested construct. The minor reason is that there might not present the recognition site for DpnI restriction enzyme in construct. The controls show that there is no digestion, and therefore DNA fragmentation was not occurred in controls. Taking all points into consideration, it can be concluded that virtual digestion shows DNA fragmentation under ideal conditions, whereas real gel shows deviations from perfect results caused by usage of different loading dye or absence of recognition site for DpnI restriction enzyme.

Reference list

1. Cheriyedath, M. S. S. Restriction Fragment Length Polymorphism (RFLP) Technique http://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx (accessed Mar 20, 2017).

2. RFLP Analysis http://www.exploredna.co.uk/rflp-analysis.html (accessed Mar 20, 2017).

3. Phillips, T. What Is the RFLP Technique? https://www.thebalance.com/rflp-definition-and-dna-analysis-applications-375574 (accessed Mar 20, 2017).