Derek Wong
Biology 275L-03
Lab 9
Restriction Enzyme acting on Plasmid and Genomic DNA
Restriction enzymes are produced by cells which are used to fragmentize DNA (deoxyribbosenucleic acid). This may function may serve as protection against foreign DNA or used in DNA regulation and is accomplished when restriction enzymes catalyze the hydrolysis of a phosphodiester linkage on each strand of DNA. There are three classes of restriction enzymes. Type I, II, and III . Type I & III have similar restriction and methylase activities and are carried about by one large enzyme complex while Type II cleave DNA at a specific recognition site and is independent of its methylase. The restriction site is where the nucleotide is cleaved. When the recognition sequence is cleaved it can create fragments that are either blunt (or flush ends), or sticky (or cohesive) ends.
Restriction Fragment Length Polymorphism is a process used to identify a DNA pattern by utilizing restriction enzymes for digestion on DNA and then using radioactive probes to create a unique “DNA fingerprint.” In this lab, restriction enzyme PstI and HindIII will be used to restrict the genomic and plasmid DNA samples isolated in the previous lab. It will also use the restriction enzyme EcoRI to create a DNA fingerprint of four given samples manifested on an agarose gel electrophoresis. There will also be a paternity test done using RFLP between a sperm donor and the suspected father.
Results
Figure 1 (see below for actual DNA picture): DNA Fingerprint of the group’s samples of genomic DNA, plasmid DNA, DNA marker and the four Suspects DNA samples. This is a depiction of the agarose electrophoresis for both groups after it has ran for 45 minutes.
Lane 1: Other group’s genomic DNA sample, Lane 2: Other group’s plasmid DNA sample (using PstI). Lane 3: Other group’s second plasmid DNA (using Hind III)
Lane 4: DNA Sample X. Lane 5:
Lane 6:
Lane 8: Standard Marker (see below for band legend)
Lane 9: Our group’s genomic DNA sample, Lane 10: Our group’s plasmid DNA sample (using PstI). Lane 11: Our group’s second plasmid DNA (using Hind III)
Lane 12: DNA Sample X. Lane 13:
Lane 14:
DNA Maker (top to bottom, bp = base pairs): 100,000 bp;
80,000 bp; 60,000 bp; 50,000 bp; 40,000 bp; 3, 000 bp; 2, 500 bp; 2,000 bp;
1,500 bp; 1, 000 bp; 750 bp; 500 bp; 250bp.
Discussion
In this lab the isolated plasmid and genomic DNA from the previous lab was exposed to restriction enzymes and digested. Four DNA samples were also exposed to restriction enzymes. These samples were then run using gel electrophoresis. The samples were then compared to the DNA marker.
For the genomic and plasmid DNA the digestion was successful because many bands could be seen, indicating digestion has taken place. Restriction enzyme digestion will yield a “smear” because there are so many bands and it will start to merge and become indistinguishable, resulting in a smear.l
The second part of the experiment involved identifying the unknown DNA sample (C) when given three known samples (X, Y, Z). This was done using gel electrophoresis. By comparing the banding pattern of the unknown sample to the known sample one can determine who the DNA belongs to (similar to identifying the unknown bacteria in lab 2). However, since the bands did not come out (for either group), it is not possible to determine who/which DNA sample is the culprit. Ideally, the banding pattern of one of the suspects will match the crime scene DNA and criminal will be found (assuming all four samples came out correctly). The samples may not have came out right because
The last part of the lab involved a paternity test involving RFLP. On paper, the DNA sequence of the child, mother, and the two potential fathers were given. Using RFLP techniques, it was determined that the biological father of the child is the sperm donor. All of the DNA involved was “cut up” into fragments and arranged from largest to smallest. Then a GTA probe was then used and a pattern could be seen. By comparing the pattern seen with the GTA (shown as CAT on the actual DNA because CAT is complementary to GTA) one can see that certain GTA sequences could only be inherited from the sperm donor (and the mother, and not the surrogate’s husband’s DNA).