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Lecture 3 (exam notes).docx

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Biopharmaceutical sciences
Linda Bonen

Lecture 3 Techniques and Tools for studying DNA Technique 1 Restriction enzymes  Cleaves DNA into specific small fragements  DNA endonucleases cut double stranded DNA at specific recognition sites (often palindromic ( same when read 5’to3’ or 3’to5’)  Recognition sequences often 4 or 6bp, but also “rare cutters” can be useful for generating very large fragments in genomic mapping  They produced blunt and sticky ends (BamHI produces sticky ends)   Exonnucleases remover nt at end of DNA/ RNA  Endonucleases make internal cuts  Two different restriction enzymes may generate same sticky ends o BamHI and Sau3AI cute 5’-3’ GATC and produc sticky ends  Isoschizomers- restriction enzymes with identical recognitions sequences  Example using isoschizomers to assess DNA methylation state of genes in cancer patients  In this study; they found that the controls had hyper methylated and hypo methylated genes (8 on the right side) but the MCL patients had reversed those methylation states (ie. The controls that had hyper methylated sites at say gene (X) the MCL patients instead had hypo methylated sites at gene (X)  Agarose Gel electrophoresis- to separate DNA fragment by size  The smaller the fragment the faster is travels, therefore it travels furthest  DNA is negatively charges and therefore loaded on the negative side and travels to the positive end  It is useful when separating out relatively smaller fragments o Pulse fiels electrophoresis is used for separation of large fragments/ whole chromosomes/megaplasmids  Pulse fiels ectrophoresis for separation of large DNA molecules  >50kb standard electro, is kinda useless  Orthogonal field alternation gell electro (OFAGE) is used- 1-2mb   But if DNA is very complex/ number of fragements is too big then you will only see continuous white bands on your gel  Why are different profules expected for genomic DNA cleaved with BamHI (6bp cutter) vs. Sau3A (4bp cutter) even though they cleave the “same” sequence? o Because probability wise it is probably going to be more common to find a 4bp sequence than a 6bp seq. o Enzyme with 6bp recognition site expected to cut a DNA molecule with 50%GC content on AVERAGE is 4^6 bp, enzyme with 4bp recog site 4^4 etc… o 2. southern hybridization  Used to detect specific restriction fragments containing sequence(s) of interest (where the sequences could be genes, and they are among all other fragments)  After electrophoresis, denature DNA and transfer it from gel to membrane so that DNA fragments remain in the same relative position  To denature DNA you can put it in an alkali solution (NaOH), it splits the DNA so that the probe can attach to one of the strands  The probe will anneal with a complimentary sequence and show as a black bar  Hybridize blot with a “PROBE” (DNA, oligomer, cDNA or RNA) which is tagged either radioactively or non radiolabelled and detect specific hybrid autoradiography  Stability of the hybrid depends on: o Length of hybrid (GC content) o Hybridization conditions (ionic strength, Temp)  Hybrid of around 50bp or longer are stable under all standard conditions   The lines are: E= 5kb S=0.5, 2, 2.5 kb  Applications for southern blots: o To identify restriction fragment carrying sequence (gene) of interest o Identify gene copy number  NOTE: northern hydribization= RNA is electrophoresed, blotted to membrane and hybridized with probe o To determine if gene is active, size of mRNA and abundance 3. PCR- polymerase Chain Reaction  To obtain one specific DNA region in large copy number  Rapid amplification of DNA region of interest by enzymatic reaction in test tube o Denature- of duplex DNA o Annealing- of 2 different primers (synthetic oligomers usually 15-25nt)- flank region of interest, in opposite orientation so anneal to opposite strands o Extension- of complimentary strand o Cycle is repeated 25-30x o Next cycle A discrete PCR product is generated, its length corresponds to distance between primers PCR in detail: 1. Target DNA mixed with taq polymerase (oligo-nt primers) 2. Heat mixture to 94C to denature DNA 3. Reduce temp to 50-60C- this allows some reattachment of DNA, but also of primers to DNA 4. Temp raised to 72C so that actual synthesis can begin 5. Then b
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