Blotting Techniques: Limitations

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• Blotting Techniques Limitations
• needs ?g amounts of DNA
• DNA needs to be relatively pure
• assay time several days to gt week

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• PCR Requirements
• heat-stable DNA polymerase
• thermocycler
• target DNA and primers

• Taq Polymerase
• Thermus aquaticus DNA polymerase
• thermophilic organism
• enzymes resistant to high temperatures
• 72-74o optimum

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PCR Protocol

• mix DNA, primers, dNTPs, Taq, buffer, Mg2
• program thermocycler for times and temps
• denaturation
• annealing
• extension
• 20-30 cycles
• analyze amplified DNA (amplicons)

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Design of Oligonucleotide Primers

• analyze sequence with computer
• amplicon length (250-1000 bp)
• uniqueness (18-28 bases)
• Tm gt 55o
• 50 GC composition
• 3'-GC 'caps'
• no internal complementarity
• no 'primer dimers'
• HPLC purification (optional)

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RNA-PCR

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

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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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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)

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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)
• Tm of probe 8-10o gt annealing temperature

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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