Dna Fragment With The Desired Gene example essay topic

#1. a) The Use of a Bacterial Plasmid to Clone and Sequence a Human Gene The process begins with restriction scanning and binding to double-stranded DNA at specific base-pair sequences, the recognition sites, in a predictable manner. The restriction sites are usually 4 to 8 base pairs long and are characterized by the palindromic sequences, with both strands having the same sequence when read in opposite direction. After the restriction endo nuclease binds, it starts to disrupt, using hydrolysis, the bonds between neighbor nucleotides, causing the H-bonds between base pairs in the cutting region to be broken. This cuts the original double-stranded DNA strand, producing two DNA fragments, which may differ for different restriction, depending on where the bond is broken when cut by the endo nuclease. This process can produce either blunt ends (where ends of the DNA fragment are fully paired with no overhangs), or sticky ends (where both DNA fragments have nucleotides lacking complementary bases and overhangs are produced).

However, sticky ends are more useful for genetic engineering. The next step, gel electrophoresis, separates the gene that has been excised, from the unwanted fragments taking advantage of chemical and physical properties of DNA. The DNA fragments travel through gel as a result of charge passed through it causing the longer fragments to separate from shorter ones, which helps in identifying gene and makes it easier to cut it out from the gel. The DNA fragment with the desired gene is, therefore, excised and purified. The same restriction endo nuclease, that is used to cut the original DNA strand, then splices this gene into a plasmid (small, circular DNA molecules found in bacteria). Because the plasmid and the foreign gene are cut by the same restriction endo nuclease, the sticky ends formed, are complementary and anneal to each other forming H-bonds.

The DNA li gase reforms the bonds, after which, the recombinant plasmid with the foreign DNA, is introduced into the bacterial cell, in the process of transformation, and replicates to form clones (exact copies of itself). Overall, this technique of altering sequence of DNA molecules became very useful for many reasons, one of which is production of hormones. Hormones such as insulin and som atropin, were produced by inserting a gene into a plasmid, and became essential hormones in medical practice. In this process, the needed human genes were incorporated into plasmids and activated or inactivated when needed, using specific inducers for promoter regions. Polymerase Chain Reaction Direct, unlike the use of bacterial plasmid, method of biotechnology to make copies of the desired sequence of DNA. At first, the DNA strands are separated using heat, which causes the H bonds to break between strands.

The two strands separate and are used as templates to build complementary strands using the Taf. polymerase (similar to DNA polymerase, but can withstand high temperatures) and two DNA primers (forward and reverse DNA primers, synthesizing DNA in opposite directions), in a process similar to DNA replication. After the complementary strands are build, the cycle repeats with new double-stranded copies of the targeted DNA build. In this method of biotechnology, however, the targeted areas on DNA is not isolated in first steps. In the first cycle, for example, only variable-length DNA strands are isolated, which start with target region DNA and extend beyond it. In the second cycle, the constant-length DNA strands are produced, which start and terminate at the target region.

After the third cycle, the number of copies of the targeted strand only increase exponentially. This cycle technique is useful in forensic criminal investigations, medical diagnostics, genetic research, and many other fields, because it requires only a small part of DNA, and takes relatively little time to accomplish. #2 a) Gel Electrophoresis is the technique used to separate a gene that has been excised, from the unwanted fragments. It takes advantage of varying length of different DNA fragments and their negative charge.

In this process, DNA fragments travel through the gel at different speeds, depending on their size. Shorter fragments travel faster, while longer fragments - slower. Electrical current is used to place negative charge at one end of the gel, where the DNA fragments are placed, while positive charge is placed at the opposite end of the gel. The electrolyte solution causes a current to pass through the gel and negatively charged DNA to migrate toward positively charged electrode. Before the process, DNA is labeled with dye containing glycerol, for easier visualization. After gel electrophoresis is complete, the gel is stained and lengths of DNA fragments are determined using the comparison experiment done using the molecular marker.

The fragment sizes can then be compared and the region of gel containing the desired fragment can be excised and purified. b) (I) The DNA digested with the enzyme X would produce one big fragment and two smaller ones. The two smaller fragments would move faster through the gel causing them to separate from the longer fragment. The two smaller fragments will be at about the same level. (II) If the DNA is digested with Y enzyme, three fragments of about the same length will be produced. Therefore when moving through the gel, they are going to more at about the same speed, causing them to be at about the same position in the gel. ( ) If the DNA is digested with both enzymes X and Y, four small fragments and one big fragment will be produced causing the four small fragments to move faster as compared to the big fragment, causing it to separate from them.

The four small fragments, though, will be at about the same level. (IV) In undigested DNA, there will be no separation as it is only one fragment. It is unable to show any variation in length until it is cut by a restriction endo nuclease. c) (I) The main purpose of the restriction endo nuclease is to cut the double-stranded DNA at specific base-pair sequence, recognition site, so that it would be easier to extract the specific gene needed from the DNA. The restrictions endo nuclease first scans the DNA strand for 4-8 base pair long recognition site, which is specific for each restriction endo nuclease. The restriction site is also palindromic, which means both strands have the same base sequence when read in opposite direction. When the restriction endo nuclease finds the restriction site, it binds to it and disrupts the bonds between the nucleotides.

This also causes the H bonds between base pairs to be broken, where cut. Depending where cut, and how the H bonds break, two types of ends can form on the resulting DNA fragments: blunt ends and sticky ends. Blunt ends are produced when ends of the fragments are fully paired; and sticky ends are produced when ends of the fragments have nucleotides lacking complementary bases. (II) If a mutation of even one nucleotide occurred at the recognition site of the Y enzyme, it will not bind to the recognition site.

This happens because of the palindromic nature of the recognition site, where even a small mutation can cause the whole sequence to be wrong and the restriction endo nuclease not to bind to it. Because there are two recognition sites for the enzyme Y, mutation may occur at either one of them or both of them. Therefore, the results of this mutation may vary. If a mutation occurs at one of the recognition sites and not the other, the enzyme will cut the DNA strand producing two fragments. If a mutation occurs at both of the recognition sites, the restriction enzyme will not be able to bind to the recognition site at all.