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Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure assay time: several days to > week specific DNA fragment(s) are.

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Presentation on theme: "Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure assay time: several days to > week specific DNA fragment(s) are."— Presentation transcript:

1 Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure assay time: several days to > week specific DNA fragment(s) are enzymatically amplified –10 6 -fold amplification possible –can detect single molecule tolerates impure DNA assay time < day Polymerase Chain Reaction (PCR)

2 Geometric Amplification 2020 2121 2 2n2n 1 st cycle 2 nd cycle n th cycle

3 Taq Polymerase Thermus aquaticus DNA polymerase thermophilic organism enzymes resistant to high temperatures 72-74 o optimum PCR Requirements heat-stable DNA polymerase thermocycler target DNA and primers

4 mix DNA, primers, dNTPs, Taq, buffer, Mg 2+ program thermocycler for times and temps –denaturation –annealing –extension 20-30 cycles analyze amplified DNA (amplicons) PCR Protocol

5 Design of Oligonucleotide Primers analyze sequence with computer amplicon length (250-1000 bp) uniqueness (18-28 bases) T m > 55 o 50% GC composition 3'-GC 'caps' no internal complementarity no 'primer dimers' HPLC purification (optional)

6 RNA-PCR aka RT-PCR make a complementary copy of mRNA use the cDNA in PCR reaction 3 basic strategies

7 Quantitative PCR titrate with known amounts of competitor laborious real time PCR use fluorescent tags and light cycler dsDNA binding dye (eg., SYBR green) specific ssDNA probes measure accumulation of product during the PCR reaction

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10 Specific RT-PCR Probes DNA-intercalating dyes are non-specific –accumulation of spurious amplicons –primer dimers and target DNA –no multiplexing ssDNA probes against amplicon add specificity –detection based upon fluorochrome and quencher pairs –hydrolysis probes (aka Taqman or F-Q probes) –molecular beacons (aka hairpin probes)

11 Taqman Probes probe contains fluorescent tag and quencher exonuclease activity of Taq polymerase releases fluorescent tag fluorescence each cycle high background from probe Primer/Probe Design 50-150 bp (amplicon) 20-26 bases (probe) T m of probe 8-10 o > annealing temperature

12 Problems and Limitations minor DNA contamination can be a serious problem need to know flanking sequences to design primers Precautions gloves filtered pipette tips sterile hood decontaminate (eg, UV) aliquot reagents add target DNA last no target DNA control prepare + control elsewhere Unknown Flanking inverse PCR add anchors use random primers RAPD AFLP


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