Restriction Enzyme Analysis of DNA
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Agarose gel electrophoresis is a method used in biochemistry and molecular biology. It is used to separate DNA or RNA molecules by size. Since DNA and RNA have negatively charge, when they go through an agarose matrix with an electric field, they will move from cathode to anode. The shorter molecules move faster and migrate farther than longer molecules; therefore the different sizes molecules can be separate. The most important factor which affects the migration is the length of the DNA molecules.
A restriction enzyme, is an enzyme that cuts double - stranded DNA following its specific recognition of short nucleotide sequences, is used to cut the DNA into small fragments. The unique recognition sequences are usually tetra- or hexanucleotide palindromes with axes of dyad symmetry. Which means the sequence on one strand reads the same in the reverse direction on the complementary strand, e.g. GTATAC and its complementary strand CATATG. Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation of the DNA fragments.
Plasmid is an extra - chromosomal DNA molecule, which is capable of replicating independently of the chromosomal DNA. It is circular and double - stranded. Plasmid is usually found in bacteria. The size of plasmid is between 1 to 200 kilobase pairs.
In this experiment, a specific recombinant plasmid pBR325 was analyzed, and hence a restriction map would be constructed.
As described in the practical manual
From the photograph, four unknown plasmid fragments migrated distance could be measured and hence the four fragments size could be found:
In lane 2: migrated distance of the fragment was 36.5mm, therefore 11.00 kilobase would be the size of this fragment;
In lane 3: migrated distance of the near fragment was 42.5mm, therefore 7.08 kilobase would be the size of this fragment; migrated distance of the further fragment was 66mm, therefore 3.98 kilobase would be the size of this fragment;
In lane 4: migrated distance of the near fragment was 40.75mm, therefore 7.94 kilobase would be the size of this fragment; migrated distance of the further fragment was 73mm, therefore 2.82 kilobase would be the size of this fragment;
In lane 5: migrated distance of the near fragment was 50mm, therefore 6.31 kilobase would be the size of this fragment; migrated distance of the further fragment was 53mm, therefore 5.62 kilobase would be the size of this fragment;
The 4 unknown plasmid size and migrated distance were showed in following table:
The size of the plasmid could be found, which was about 11 kilobase. Since the plasmid was only cut by EcoRI + BamHI in about 4:8 ratio of the plasmid, therefore the restriction map for EcoRI + BamHI could be construted:
The nucleic acids migrated from cathode to anode since it had negatively charge.
The cutting ratio of three enzymes could be found by the size of fragment as result showed. The last lane, which was the uncut plasmid, was loaded, in order to compare with the EcoRI enzyme cut fragment. The migrated distance of this uncut plasmid was large than EcoRI cut's migrated distance, which could be measured from graph. EcoRI cut's fragment was like uncoiled elastic. It encounters more resistance migrating through a gel because it is spread out and will be in direct contact with more of the gel matrix. Uncut plasmid was tightly coiled, like a balled up elastic. While the molecule may be the same size, the coiling compresses it, allowing it to encounter less resistance when migrating through the gel. This indicated the minimum base pairs of the plasmid.
If a hybrid recombinant plasmid was constructed from pBR325 by the insertion of a fragment of DNA at the BamHI restriction site, firstly the total size of the plasmid got bigger. To determine the size of the inserted fragment, EcoRI and PstI were used to cut the plasmid, and result was compared with original plasmid's fragment to get the size of the inserted fragment.
There was no additional band visible behind the main uncut plasmid band in lane 7. If there ware, they must be the chromosomal DNA bands, the absent of these in lane 2, 3, 4 and 5 was because they were too small, might not be seen, or ran off the gel.
The fluorescent bands of the DNA restriction fragments in lane 2, 3, 4, and 5 were several - fold brighter than the uncut plasmid band in lane 7. It was because that the DNA fragment are free at both ends and can 'uncoil' and pick up as much EtBr as it fits, whereas the uncut plasmid could not uncoil more than a certain amount without the phosphate chain breaking: for every incorporated molecule of EtBr it had an area of local 'under-coiling' that had to be compensated by another area of 'over-coiling', this area would not incorportate any EtBr. So the number of EtBr molecules it could pick up was limited because of sterical considerations. Therefore the DNA fragment had less sterical restrictions, stained more than the uncut plasmid.
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