Lecture-5

Lecture-5 - #5 Recombinant DNA technology(II 1 DNA Analyses...

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#5 Recombinant DNA technology (II) 1. DNA Analyses 2. RNA analyses
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1. DNA analyses How to rapidly clone a gene? What is the nucleotide sequence of a gene? How to rapidly distinguish genomes of two individuals? Where is a gene in the chromosome? Methods used to analyze DNA : 1. PCR 2. Southern blot and DNA fingerprint 3. DNA sequence
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(1) Polymerase Chain Reaction (PCR) A first generation PCR machine PCR REACTION : multiple cycles of the denaturation/renaturation/DNA synthesis reaction to amplify a specific piece of DNA. In addition to the DNA template, a PCR reaction also needs: PRIMERS : Oligonucleotide primers that span the region to be amplified. So, one has to know the DNA sequence of the target DNA to amplify it using PCR TAQ POLYMERASE : it is a heat-stable DNA polymerase isolated from Thermus aquaticus (an Archae living in hot spring). Taq polymerase can survive extremely high temperature that denatures most enzymes. PCR is an in vitro (i.e. out of the cell or in the test tube) DNA amplification reaction, originally invented by Kary Mullis in 1983 (Noble price 1993).
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A PCR reaction has many cycles of the same process The process is usually repeated 25-35 cycles. Each cycle has three steps: denature, annealing, and elongation/DNA polymerization . More than 1 billion copies of DNA can be amplified with 35 cycles of PCR reaction from a single copy of DNA 95 o C/55 o C 72 o C 95 o C/55 o C 72 o C 95 o C/55 o C 72 o C
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1. PCR cloning : a genomic DNA or a cDNA can be easily amplified and cloned 2. RT-PCR/Q-PCR : reverse-transcription (RT) products of different RNA samples can be PCR amplified and compared 3. Disease diagnosis : trace amount of pathogens (e.g. HIV) can be amplified and detected 4. Forensics : DNA of samples collected from the crime scene can be amplified and compared with that of a suspect 5. Evolutionary study : DNA from extinct species can be obtained and analyzed PCR is a very useful technique
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-Isolate genomic DNA -Anneal to primers spanning the region that needs to be cloned. The primers contain a restriction site found in the MCS -PCR reaction -Cut and clone into a vector -using PCR, one can clone a piece of DNA without a DNA library. PCR cloning (I) PCR Restriction digestion primers complementary to the sequence of the DNA to be cloned
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It has been found that Taq DNA polymerase always adds a single A to the 3' ends of its product, so PCR amplified DNA contains 3’ A-overhangs. The A-overhang DNA can be cloned into a vector containing a T-overhang. This vector, or T-vectors could be prepared by tailing a single T residue at both 3’ ends of a linearized and blunt-ended plasmid with terminal deoxynucleotidyl transferase , or it may be prepared by restriction enzymes such a XcmI . This method is called TA cloning PCR cloning (II) XcmI digested vector Any PCR product 3' 3' 5' 5' A A
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Lecture-5 - #5 Recombinant DNA technology(II 1 DNA Analyses...

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