6.3 Advanced Molecular Biological Techniques
1. Polymerase chain reaction (PCR)
2. Restriction fragment length polymorphism (RFLP)
3. DNA sequencing
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
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
Polymerase Chain Reaction (PCR)One cycle:1. Double-stranded DNA is denatured using
heat (94oC–96oC) 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 (50oC–65oC)
• No RNA primer
3. Taq polymerase add complementary nucleotides to synthesize the new DNA strand (72oC)
• 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
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
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
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
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
RFLP Analysis4. Southern blotting:
i. Transfer DNA from gel to nylon membraneii. 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::Southern Blot
RFLP analysis Differences in
pattern to detect polymorphisms
Animation
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
Sanger dideoxy methodInto 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)
Sanger dideoxy method If dideoxy analogue is missing 3’-OH
on the deoxyribose sugar, DNA polymerase cannot add the next complementary basesynthesis 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
Sanger Method Animation
http://www.mefeedia.com/watch/21777157
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
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.
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
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
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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