Advanced Microbiological Techniques

1. Advanced Microbiological Techniques • The Polymerase Chain Reaction (see Fig. 1, P. 297) • (an advanced method of amplifying DNA to prepare large samples for analysis) • proposed by Kary Mullis in 1987, closely related to DNA replication • normally, genes are cloned by insertion into a plasmid, then harvested by extraction of the plasmid and excision of the gene fragment •  the polymerase chain reaction is a direct method of making copies of a gene sequence • heat (vs. gyrase/helicase) is used to separate double helix and both strands are used as templates to build complementary strands • DNA primers (vs. RNA) are short nucleotide sequences complementary to the 3’-5’ ends of the template DNA on each opposing strand (5’-3’ primers) •  forward and reverse DNA primers synthesize DNA in opposite directions toward each other • Taq polymerase (isolated from thermophilic bacterium Thermus aquaticus) allows for the building of complementary strands at relatively high temperatures (72oC, vs. DNA polymerase III which denatures at 37oC) •  template and complementary strands are separated and primers annealed to repeat the cycle  doubling of double-stranded copies of target area results in exponential increase (2n  i.e. 230 > 1 000 000 000)

2. target area is not completely isolated in the first few cycles of DNA replication • First Cycle: two variable-length strands are produced that start at the target region where the primer has annealed on one end, and extend beyond the target region on the other end •  Second Cycle: on two of the strands, one end terminates at the target region (from the variable-length strands produced in the first cycle), and the primers anneal to the other end of the target area •  nucleotides are added, commencing from the primer and ceasing when the end that terminates at the target region is reached, resulting in constant-length strands •  other two strands are extended by Taq polymerase as in the first cycle • .third cycle: target strand copies increase exponentially • Use of PCR • forensic criminal investigations (small amount of DNA needed as starting point for copying) • medical diagnosis (i.e. instead of waiting for presence of HIV antibodies, primers are designed that are complementary to HIV DNA, then amplified to look for HIV genome) • genetic research (species relatedness from fossil evidence)  Practise – P. 298, #1,2

3. Restriction Fragment Length Polymorphism (an advanced method to sort and analyze DNA molecules) • individuals of the same species have different alleles for the same genes, so their genome is polymorphic •  polymorphisms in coding regions can identify individuals with mutations •  in noncoding regions, variations exist in the number of variable number tandem repeats (VNTR’s)or microsatellites (repeating base pair sequences varying in length and position) which can be exploited in forensic investigations • gel electrophoresis is unable to resolve individual fragments because they are too numerous and differ only slightly in size over a wide range •  RFLP analysis compares lengths of DNA fragments produced by restriction enzyme digestion and revealed by complementary radioactive probes to determine genetic differences between individuals • (refer to Figure 3, P. 299)

4. double-stranded DNA is denatured to become single-stranded, then transferred and bound to a nylon membrane (negatively charged DNA is “blotted” onto membrane by placing a positive electrode behind the gel = Southern blot) single-stranded DNA is immersed in a solution containing radioactive complementary nucleotide probes for specifically chosen regions (regions may be mutations in an allele or a variable number tendem repeat in a noncoding region) • hybridization produces complementary base pairing between the probes and the DNA the nylon membrane is placed against X-ray film, where radioactive probes cause exposure in the areas of hybridization and produces an autoradiogram  the pattern revealed in the exposed film can be used to match a suspect’s DNA to the DNA found at a crime scene Practise – P. 300, #6-14

5. DNA Sequencing (refer to Fig 4, P. 301) • the Sanger dideoxy (chain termination) method is the the most popular and easily automated DNA sequencing protocol • dideoxy analogs of deoxynucleoside triphosphates (dNTPs) are missing the OH-group from the deoxyribose sugar DNA polymerase is unable to catalyze the phosphodiester linkage between the growing chain and the incoming nucleotide • A radioactive DNA primer is annealed to a single-stranded DNA template • Identical copies of the primed template are placed in four separate reaction tubes, each containing DNA polymerase, deoxynucleoside triphosphates (dATP, dTTP, dGTP, dCTP) plus a radioactively labelled dideoxy analogue (ddNTP) of one of the dNTPs • since only a fraction of the dNTPs are dideoxy analogues, different lengths of complementary DNA will be built before the analogue is incorporated and the chain stopped • Contents of all four reaction tubes are loaded into separate lanes of a gel and the strands separated by electrophoresis •  DNA sequence can be read directly from the gel in ascending order (see Figure 6, P. 302)