1. 6.3 Advanced Molecular Biological Techniques 1. Polymerase chain reaction (PCR) 2. Restriction fragment length polymorphism (RFLP) 3. DNA sequencing

2. Polymerase Chain Reaction (PCR) 4.4.1: Outline the use of polymerase chain reaction (PCR) to copy and amplify minute quantities of DNA. [Obj. 2] Until the late 1980s, many copies of a desired DNA fragment could only be made by inserting the DNA sequence into plasmids Problem: The plasmids had to be extracted from bacteria, and then the desired DNA fragment had to be excised Solution: Direct method of making copies of a desired DNA sequence, called polymerase chain reaction (PCR) – Kary Mullins, 1985

3. Polymerase Chain Reaction (PCR) PCR: Amplification of DNA sequence by repeated cycles of strand separation and replication Small sample of DNA can be amplified to make multiple copies of a desired DNA fragment Each PCR cycle doubles the copies of a desired DNA fragment, resulting in exponential growth –ie. after 30 cycles, > 1 000 000 000 copies (2 30 ) are made http://users.ugent.be/~avierstr/principles/pcrcopies.gif

4. Polymerase Chain Reaction (PCR) One cycle: 1. Double-stranded DNA is denatured using heat (94 o C – 96 o C) to separate strands by breaking hydrogen bonds No DNA helicase or DNA gyrase 2. DNA primers (5 ’ -3 ’ ) anneal to complementary template DNA that bracket the desired DNA sequence (50 o C – 65 o C) No RNA primer 3. Taq polymerase add complementary nucleotides to synthesize the new DNA strand (72 o C) No DNA polymerase III Repeat cycle (steps 1-3) http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html http://croptechnology.unl.edu/animationThumbnails/1020458324.gif

5. PCRPCR: Length of DNA strands Targeted DNA sequence is not completely isolated in the first few cycles of PCR Variable-length strands: Mixture of replicated DNA strands of unequal length –After first cycle, variable-length strands start at target region on one end and extends beyond the target region on the other end Constant-length strands: Mixture of replicated DNA strands of equal length –After second cycle, two of the replicated strands start at target region on one end and terminates at target region on the other end –By third cycle, number of copies of targeted DNA strands increases exponentially

6. www.maxanim.com/genetics/PCR/PCR.htm http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535: :535::/sites/dl/free/0072437316/120078/micro1 5.swf::Polymerase Chain Reaction

7. Restriction Fragment Length Polymorphism (RFLP) Polymorphism –any difference in DNA sequence (coding or non- coding) that can be detected between individuals Restriction Fragment Length Polymorphism Analysis –technique that compares different lengths of DNA fragments produced by restriction endonucleases to determine genetic differences between individuals by using complementary radioactive probes http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/007243731 6/120078/bio20.swf::Restriction Fragment Length Polymorphisms

8. Restriction Fragment Length Polymorphism Analysis 1. Digest DNA using restriction enzyme(s) 2. Run digested DNA on gel using gel electrophoresis Smear - Many DNA fragments with slight differences in length 3. Expose gel to a chemical to denature double-stranded DNA to become single-stranded 4. Southern blotting

9. RFLP Analysis 4. Southern blotting: i.Transfer DNA from gel to nylon membrane ii.Expose nylon membrane to solution with radioactive complementary nucleotide probes that hybridize to specifically chosen DNA sequences on nylon membrane iii.Place nylon membrane against X-ray film, where hybridized radioactive probes cause exposure of X-ray film, producing an autoradiogram http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535: :/sites/dl/free/0072437316/120078/bio_g.swf::Southe rn Blot

10. RFLP analysis  Differences in pattern to detect polymorphisms Animation

11. DNA Sequencing Determine sequence of base pairs for genes Sanger dideoxy method – DNA sequencing technique based on DNA replication using dideoxynucleoside triphosphate http://www.sanger.ac.uk/I nfo/Intro/gfx/fred_bw.jpg

12. Sanger dideoxy method Into 4 reaction tubes, add: Double-stranded DNA to be sequenced is denatured to become single-stranded Radioactively labelled primer to end of the DNA template DNA polymerase Free nucleotides (dATP, dTTP, dGTP, dCTP) Into each of the 4 reaction tubes, add a different radioactively labelled dideoxy analogue (nucleoside triphosphate that has no hydroxyl group on the 2 ’ and 3 ’ carbon of ribose sugar)

13. Sanger dideoxy method If dideoxy analogue is missing 3 ’ - OH on the deoxyribose sugar, DNA polymerase cannot add the next complementary base  synthesis stops Chain termination resulting in different DNA fragment lengths Separate different DNA lengths by gel electrophoresis, loading each reaction tube in a separate well/lane Sequence can be read from the gel in ascending order http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

14. Sanger Method Animation http://www.mefeedia.com/watch/217771 57 http://www.mefeedia.com/watch/217771 57

15. Human Genome Project To determine the genetic sequence of the 46 human chromosomes Used similar sequencing technique, but used fluorescently tagged ddNTPs that could be read by a computer

16. 4.4.6: Outline three outcomes of the sequencing of the complete human genome. [Obj. 2] It is now easier to study how genes influence human development. It helps identify genetic diseases. It allows the production of new drugs based on DNA base sequences of genes or the structure of proteins coded for by these genes. It will give us more information on the origins, evolution and migration of humans.

17. 4.4.11: Define clone. [Obj. 1] Clone: a group of genetically identical organisms or a group of genetically identical cells derived from a single parent cell. http://www.dnalc.org/resources/animations/cloning10 1.html http://www.dnalc.org/resources/animations/cloning10 1.html http://learn.genetics.utah.edu/content/tech/cloning/cli ckandclone/ http://learn.genetics.utah.edu/content/tech/cloning/cli ckandclone/ Steps for cloning a gene: http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/si tes/dl/free/0072437316/120078/micro10.swf::Steps in Cloning a Gene

18. 4.4.12: Outline a technique for cloning using differentiated animal cells. [Obj. 2] http://www.massasoit- bio.net/courses/136/136_courseassets/cummings_animations/cloning.html

20. 4.4.13: Discuss the ethical issues of therapeutic cloning in humans. [Obj. 3] Source: http://www.ibguides.com/biology/notes/genetic-engineering-and-biotechnology