Chapter 12: DNA Manipulation

Restriction enzyme: an enzyme that cleaves DNA at a specific recognition nucleotide sequence (restriction site/sequence), e.g. Eco R1, Hap II. Naturally occur in bacteria and archaea. Different types can produce either blunt ends or sticky ends.

Electrophoresis: technique for sorting through a mixture of DNA fragments through an electric field on the basis of different fragment lengths. 1. DNA fragments are placed at one end of a gel. 2. An electric field is applied to the gel, with the + at the far end and the at the DNA end. 3. Due to the net - charge of DNA (as a result of the phosphate groups), the fragments will move towards the + end. 4. Smaller fragments move quicker than larger ones, thus the end result will show bands of DNA at different distances down the gel. 5. These are compared with bands of DNA fragments of known length.

Probe: a single-stranded segment of DNA or RNA with a base sequence complementary to that of a target strand of DNA, carrying a radioactive or fluorescent label on it. The target DNA must be denatured so the probe can attach to the target DNA sequence. Hybridisation: formation of hydrogen bonds between single-stranded complementary DNA segments, e.g. when a probe attaches to its target DNA sequence. In situ: in place, in situ hybridisation occurs when a probe hybridises to DNA that is in its usual place in the chromosomes of a cell.

Ligase: enzyme that catalyses the joining of two double-stranded DNA fragments. Ligase forms covalent bonds at the sugar-phosphate backbones of DNA fragment. Ligase is also involved in the joining of Okazaki fragments in DNA replication.

Vector: in genetics, an agent that carries passenger DNA into a cell. 1. 2. 3. 4. 5. 1. 2. Plasmid: a small, circular fragment of extra-chromosomal DNA present in bacterial cells. DNA of plasmids is cut at one point with a restriction enzyme that produces sticky ends. Passenger DNA is cut at both ends with the same restriction enzyme. Plasmid and passenger DNA are mixed; their sticky ends join by hydrogen bonding. Ligase joins the sugar-phosphate backbones of the DNA fragments. The plasmids with the passenger DNA can be reintroduced to the bacterial cell. Plasmids are reintroduced to the bacterial cell through an electric pulse, or a heat shock. Positive cations surround the cell to attract the plasmids (with a - charge). A sudden rise in temperature (heat shock) weakens the cell wall and membrane and allows plasmids to enter the cell. Viruses can also be vectors.

Synthesising specific DNA DNA synthesiser: the instrument joins nucleotides in a predefined order of base sequences to produce primers or probes. Reverse transcription: the enzyme reverse transcriptase uses mature mRNA as a template and creates a single-stranded DNA with complementary base sequence, it is known as cDNA (complementary DNA). o DNA polymerase can make it double-stranded. o Thus DNA without introns can be created, allowing eukaryotic genes to be inserted into prokaryotes.

Gene cloning: process of making multiple identical copies of a specific gene or segment of DNA. 1. 2. 3. The gene to be cloned is put into a plasmid, which is re-inserted to a bacterial cell. Within the bacteria, the plasmid multiplies to up to 20 copies. Binary fission of the bacteria every 20 minutes increases the number of plasmids with the gene. Human genes in bacteria can be expressed to produce the proteins, e.g. produce Humulin (insulin).

Polymerase chain reaction (PCR): technique for amplifying small amounts of DNA exponentially through many cycles of multiplication. 1. 2. 3. 4. PCR is carried out in vitro (in a test tube). DNA is heated to 94C so it denatures into single strands. DNA primers bind to the ends of each DNA strand at 55C. Taq polymerase enzyme adds nucleotides to the primer at 72C. Repeat many times. Each cycle takes about 5 minutes. Taq polymerase is a bacterial enzyme which operates at higher temperatures than human DNA polymerase. Thus it would not denature at the temperatures required to denature DNA. Other methods of amplifying DNA are slower and less convenient than PCR.

DNA sequencing: techniques for determining the sequence of nucleotide bases in a strand of DNA. 1. 2. 3. One DNA sequencing method is known as the dideoxynucleotide or Sanger method. The DNA is amplified and then heated to denature and separate the double strands. A radioactive primer binds to the beginning of the section of DNA to be sequenced. Nucleotides are added with a polymerase. One of the nucleotides has an OH group removed (a dideoxynucleotide). No more nucleotides can be added after a dideoxynucleotide. 4. This is done four times each with a different dideoxynucleotide (G, T, A, C). 5. This results in segments of DNA of different lengths. 6. Gel electrophoresis then separates the segments according to size. The DNA sequence can then be deduced by examining relative positions of the DNA fragments of different dideoxynucleotides.

Gene transfer between species Transgenic organisms (TGOs): organisms that carry in their genomes one or more genes artificially introduced form another species. o Transfected: describes eukaryotic cells which contain foreign DNA in their genomes. o Transformed: describes prokaryotic cells which contain foreign DNA in their genomes.

Gene transformation: the process of putting functional recombinant DNA into a bacterial cell. Earliest method: micro-injection of DNA into the nucleus of a fertilised egg of the second species. o However the gene was randomly incorporated into chromosomes; it may be integrated in the middle of an essential gene. Newer methods: using bacterial plasmids/viruses/adenoviruses as vectors to insert the gene.

DNA in identification DNA fingerprinting: technique of identifying DNA of individuals, based on minisatellites, using multi-locus probes. - DNA profiling: technique of identifying DNA of individuals, based on STRs/microsatellites, using a number of single-locus probes. - Restriction fragment length polymorphism (RFLP): - Minisatellites (also variable number tandem repeats): chromosomal sites at which sequences of 10-60 base pairs are repeated. - Microsatellites (also short tandem repeats): chromosomal sites at which sequences of 2-4 base pairs are repeated. - Hypervariable regions (HVRs): regions in the noncoding region of mtDNA that vary greatly between unrelated individuals. Individuals from the same maternal line have identical mtDNA profiles. 1. STRs/VNTRs are cut from chromosomal DNA using a restriction enzyme and separated by gel electrophoresis. 2. The DNA fragments are transferred to a membrane using Southern blotting. 3. The DNA fragments are exposed to a single-locus/multi-loci probe carrying a radioactive label. - DNA profiling is more sensitive and faster than DNA fingerprinting, and requires smaller amounts of DNA.