LIFESCI 23L Lecture Notes - Lecture 5: Dna Profiling, Thymidine Triphosphate, Reverse Transcription Polymerase Chain Reaction
Lecture E: DNA Isolation
OVERVIEW OF SKILLS CONCEPTS
● 3-part lab
○ Isolate cheek cells in DNA and prepare for PCR
● Skills: Micropipetters, Chelex extractions of DNA from cheek cells, and using BLAST online bio-
informatics program to check primer for uniqueness
● Concepts: primer design, interpreting primer 3 results, mitchiondrial liniage, how primers work from 5-3’
BACKGROUND
● PCR: polymerase chain reaction
○ Can isolate and amplify target DNA from original DNA template; amplified DNA can then be
sequenced
○ Many applicatins!
○ Requirements:
■ Template: DNA of interest
■ Pair of synthesized primers → must lie on either side of target DNA
■ Enzyme to carry out amplification (DNA polymerase) → will use Taq (heat resistant)
polymerase
■ Four nucleotides: dATP, dGTP, dCTP, dTTP
○ Steps
■ 94* → melting → DNA denatures, strands come apart
■ 50* → annealing → primers bind to DNA
■ 72* → elongation → taq polymerase builds opposite strand using tempalte DNA
■ 94* → melting begins again, cycle starts again
● 30 cycles give us about 1 billion fold amplification (theoretical maximum as PCR
is not always completely efficient)
○ Applications
■ Genotyping: detecting different alleles of interest in an individual
● Small sample collected, DNA is isolated and amplified w/ PCR, then run through
gel
■ Diagnostics
● RT-PCR:
● Viral RNA from HIV is converted to cDNA (RT), which is then used as template
for PCR
● Blood taken from patient, if HIV present, RT will convert RNA to cDNA, which will
be then used as a template for PCR amplification
○ → used to diagnose HIV!
■ Gene cloning
● Can be used to add sequences to a DNA fragment!
○ Primer pair contains dna comlementary to template DNA AND additional
sequences that will be added during elongation
■ Amplified product will have target DNA and additional sequence
■ Good for transformation w/o restriction sites
■ PCR in foresnics
● DNA fingerprinting!
○ Humans have regions have high variability: polymorphic regions
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Isolate cheek cells in dna and prepare for pcr. Skills: micropipetters, chelex extractions of dna from cheek cells, and using blast online bio- informatics program to check primer for uniqueness. Concepts: primer design, interpreting primer 3 results, mitchiondrial liniage, how primers work from 5-3". Can isolate and amplify target dna from original dna template; amplified dna can then be sequenced. Pair of synthesized primers must lie on either side of target dna. Enzyme to carry out amplification (dna polymerase) will use taq (heat resistant) polymerase. 94* melting dna denatures, strands come apart. 50* annealing primers bind to dna. 72* elongation taq polymerase builds opposite strand using tempalte dna. 94* melting begins again, cycle starts again. 30 cycles give us about 1 billion fold amplification (theoretical maximum as pcr is not always completely efficient) Genotyping: detecting different alleles of interest in an individual. Small sample collected, dna is isolated and amplified w/ pcr, then run through gel.
