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6.3 Advanced Molecular Biological Techniques

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

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6.3 Advanced Molecular Biological Techniques

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  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 (230) are made http://users.ugent.be/~avierstr/principles/pcrcopies.gif

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

  5. PCR: 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/micro15.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/0072437316/120078/bio20.swf::Restriction Fragment Length Polymorphisms

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

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

  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/Info/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/21777157

  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/cloning101.html • http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/ • Steps for cloning a gene: • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/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

  19. 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

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