Transcript Alu-TPA PCR Kit (#8) Tech Service Training August ‘99

The Search for a
Jumping Gene:
Use PCR to Determine Your
Molecular Genotype
AdaptedModule
by Dan Murray
from aon
presentation
by:
based
a kit from
Stan
Hitomi - Monte
Vista High School, Danville,
Bio-Rad
Laboratories,
Inc. CA.
Kirk
Brown - Tracy High School, Tracy, CA.
Outline
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What is PCR?
PCR Mechanism
Transposable Elements
PV92 PCR
Experimental Protocol
Expected Results
What is PCR?
What is PCR?
• PCR stands for Polymerase Chain
Reaction
• The technique was invented by Kary
Mullis in 1983. Awarded the Nobel
Prize in Chemistry in 1993.
What is PCR?
• DNA replication
gone crazy in a tube!
• Only a small region
of the DNA actually
gets replicated
What’s in a PCR Reaction?
• Source DNA – contains target DNA region to be
copied
• Primers – short single stranded DNAs
complementary to ends of target DNA region
• Nucleotides – dATP, dCTP, dGTP, dTTP; the
building blocks of DNA strands
• Taq DNA polymerase – temperature resistant
enzyme which builds DNA strands
PCR Mechanism
How does PCR work?
• Mix PCR reaction reagents
• Cycle through various temperatures
• 94oC – Melting
• ~55oC – Annealing
• 72oC – Extension
• Repeat
Melting: 94ºC
Heat separates strands
5’
3’
3’
5’
94oC
5’
3’
3’
5’
Annealing: ~55ºC
Primers bind template
5’
3’
3’
5’
~55oC
5’
3’
3’
5’
3’
3’
5’
5’
Extension: 72ºC
Taq polymerase builds strands
3’
5’
3’
5’
3’
3’
5’
5’
72oC
5’
3’
3’
5’
3’
3’
5’
5’
Desired-length PCR product made
starting in third cycle
Cycle 1
5’
3’
5’
5’
3’
5’
Cycle 2
3’
5’
3’
Cycle 3
3’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
Transposable Elements
What are Transposable
Elements?
• Segments of DNA which have the ability to
move to or be copied to other regions of the
genome
Replicate
are
thought
Element
• Generally, they
of as “Selfish DNA”;
they usually neither benefit nor harm their host
• Discovered by Barbara McClintock (1940s-50s)
while working with maize
Donor
molecule
Recipient
molecule
Alu Elements
• Alu elements are a type of transposable element
• Alu elements are 300bp in length
• Found only in primates (including humans)
• Approx. 500,000 Alu copies per haploid genome,
representing about 5% of the genome!
• Named for the Alu I restriction site within the
element
Alu at PV92
• PV92 is a locus on human chromosome 16
• An Alu insertion occurred there within the
last 1,000,000 years
• Possible Genotypes at PV92: +/+ +/- -/-
• Used in population genetics, paternity
analysis, and forensics
• Not associated with any phenotype
PV92 PCR
The Target DNA
Chromosome
16
PV92
Locus
Alu
Element
Some Got It, Some Don’t
PV92
with
Alu
PV92
without
Alu
PCR
PCR
Experimental Protocol
The Procedures in a Nutshell
• Isolate DNA from cheek cells
• Build PCR reaction
• Do PCR in thermal cycler
• Analyze using agarose electrophoresis
Isolation of Cheek Cell DNA
•Collect cheek cells and place in
InstaGene Matrix
•Matrix binds cellular Mg2+
•Agitate
•Disperses cells in Matrix
Isolation of Cheek Cell DNA
56°C
Loosens connective tissue
and inactivates nucleases
100°C
Ruptures cell membranes
and denatures proteins
Isolation of Cheek Cell DNA
Gets rid of
InstaGene Matrix
Target DNA
now ready!
Assemble PCR Reaction
Add PCR Master
Mix containing:
•Nucleotides
•Primers
•Reaction buffer
•Electrophoresis
dyes
•Taq polymerase
PCR reaction now
ready to go!
Amplify DNA with PCR
Controls will show the three
possible outcomes
Load, Run, and Stain Gel
Stain gels and analyze.
Expected Results
PV92 Amplification Specifics
Primer binding sites
No Alu:
Region to be amplified
With Alu:
Now assume an Alu element has inserted at that locus
Alutoelement
Region
be amplified
PV92 Amplification Specifics
No Alu:
641bp
Region to be amplified
With Alu:
941bp
300bp
Region to be amplified
Actual Results
+/+ -/- +/-
941 bp
641 bp