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Lecture

6. Molecular Genetics Techniques I.pdf

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Department
Biology (Sci)
Course
BIOL 200
Professor
Richard Roy
Semester
Fall

Description
Naveen Sooknanan McGill Fall 2011 Molecular Genetic Techniques: DNA Cloning, Polymerase Chain Reaction (PCR) and DNA Sequencing are the fundamental techniques used in molecular biology. Other, more complex techniques borrow general aspects from these 3 techniques.  For many reactions involving DNA in molecular biology, a large amount of DNA is required, which can be difficult to obtain without the use of mainly DNA cloning and PCR. Plasmids are circular, double stranded DNA molecules (dsDNA) which are much smaller than genes (usually 2000-10000 kbp)  Plasmids are the most common vectors used in DNA technology o Vectors are media which carry specific genes of interest  They are extrachromosomal, meaning they are not part of the genome  They can be found in bacteria (mainly prokaryotes) and lower eukaryotes  The replication of plasmids occurs before cell division The E. coli plasmid is a common vector used in labs.  The polylinker contains multiple endonuclease restriction sites which scientists can cleave and genes of interest can be inserted.  The amp gene codes for ampicillin resistance o ampicillin is an antibiotic which normally kills E. coli  The ORI is the origin of replication for this plasmid (prokaryotes only have one origin) DNA cloning involves the insertion of a desired DNA fragment into a plasmid and allowing the replication procedure to take place, producing many more copies of the desired gene.  A restriction endonuclease cleaved the phosphodiester bonds at the restriction site o There is only one restriction site per endonuclease on one plasmid  This breaks the circular plasmid into a linear DNA molecule  Although restriction endonucleases normally create cuts with sticky ends, Sma1 is able to cleave straight through the DNA molecule, creating blunt ends  The vector DNA molecule is then able to be attached to the plasma fragment with a corresponding sticky ends o This is carried out by an enzyme carried out by T4 DNA ligase using 2 ATP and producing 2 AMP and 2 Pyrophosphate  This creates a recombinant plasmid (meaning it contains foreign DNA)  The recombinant plasmid is then mixed with E. coli and treated with CaCl a2d 42° C heat pulses to make the cell membrane permeable, allowing the plasmid to enter the E. coli cells (transformation)  The cultures are then placed on agar plates containing ampicillin which allows differentiation of only the transformed cell 1Naveen Sooknanan McGill Fall 2011 o Wild type cells do not have ampicillin resistant plasmids die when they are placed on the plate o Transformed cells survive due to the presence of the Amp gene in the plasmids  Cell division and plasmid replication both increase the amount of plasmid vectors in the colony o These cells form a colony of recombinant cells with spatial limitations o Plasmid replication is independent of chromosome replication Plasmids nowadays are heavily engineered to replicate more quickly than naturally found plasmids. Although they vaguely resemble naturally found plasmids, the plasmids commonly used in labs are synthetic and cannot be found naturally.  Using this method, it is possible to create a library of DNA fragments which can all be retrieved with one endonuclease The polymerase chain reaction (PCR) is a very fast procedure of duplicating DNA and can be done in 25 minutes to 2 hours, compares to the 3-4 days it takes for DNA cloning to take place. PCR requires:  A DNA template to polymerize, part of genomic DNA  Oligonucleotide primers (DNA primers) which can be purchased given that the DNA being polymerized is already known  Taq DNA polymerases, which are takes from a species which lives in extreme temperature conditions o Taq DNA polymerase can withstand the heat involved with PCR  dNTPs used for DNA synthesis  A thermal cycler which is capable of creating the heat patterns necessary in a PCR reaction An important aspect of DNA is its ability to denature when heated but more importantly, its ability to renature when cooled back down. This is a fundamental principle exploited in PCR. A cycle of PCR involves  First, heating the DNA to 95° to denature the DNA o This heat will denature most DNA molecules  Second, cooling to 60° to anneal primers o The unwound template due to the high temperature allows for the free binding of oligonucleotide primers  And third, heating to 72° to promote DNA extension o dNTPs and DNA polymerases present in excess to speed up the reaction  This cycle is repeated multiple times (usually 20-40 times) in order to increase the amount of DNA produced exponentially The synthesis of new duplexes proceeds very rapidly in PCR. Starting with one duplex, approximately 2 duplexes are produced after n cycles.
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