Restriction Enzyme Analysis of DNA

Restriction Enzyme Analysis of desoxyribonucleic acidAgarose gel electrophoresis is a method use in biochemistry and molecular biology. It is used to separate deoxyribonucleic acid or ribonucleic acid molecules by sizing. 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 blistering and migrate farther than longer molecules because the different sizes molecules can be separate. The most important cistron which affects the mig balancen is the length of the DNA molecules.A restriction enzyme, is an enzyme that cuts double stranded DNA following its specific scholarship of short nucleotide sequences, is used to cut the DNA into small take aparts. 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 puff direction on the complementary strand, e.g . GTATAC and its complementary strand CATATG. Recognition sequences in DNA differ for severally 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 put in in bacteria. The size of plasmid DNA 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.METHODAs described in the practical manualRESULTSFrom the photograph, quatern unknown plasmid fragments migrated quad could be deliberate and hence the four fragments size could be foundIn bridle-path 2 migrated space of the fragment was 36.5mm, therefrom 11.00 kilobase would be the size of this fragmentIn 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 fragmentIn 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 fragmentIn 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 fragmentThe 4 unknown plasmid size and migrated distance were showed in following tableThe size of the plasmid could be found, which was ab place 11 kilobase. Since the plasmid was only cut by EcoRI + BamHI in about 48 ratio of the plasmid, therefore the restriction map for EcoRI + BamHI could be construtedDISCUSSIONThe nucleic acids migrated from cathode to anode since it had negatively charge.The cutting ratio of th ree enzymes could be found by the size of fragment as result showed. The last lane, which was the raspy plasmid, was loaded, in order to compare with the EcoRI enzyme cut fragment. The migrated distance of this uncut plasmid was large than EcoRI cuts migrated distance, which could be measured from graph. EcoRI cuts fragment was like uncoiled elastic. It diddles 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 launching of a fragment of DNA at the BamHI restriction site, firstly the total size of the plasmid got bigger. To hold back the size of the inserted fragment, EcoRI and PstI were used to cut the plasmid, and result was compared with original plasmids 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 bill without the phosphate chain breaking for every incorporated molecule of EtBr it had an area of local under-coiling that had to be even out 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 consid erations. Therefore the DNA fragment had less sterical restrictions, stained more than the uncut plasmid.