PCR

(polymerase Chain Reaction)

 Alu PCR Kit
•Introduction
•Extract genomic DNA and prepare PCR
samples
•Cycle samples
•Agarose gel analysis
•Hardy-Weinberg analysis
 The Polymerase Chain
Reaction
• What is PCR?
• What is needed for PCR?
• How does PCR work?
• What are we amplifying?
What is PCR?
•DNA replication gone crazy in a
tube!
•Makes many copies target
sequence from template
•Uses Taq Polymerase heat-resistant
DNA polymerase from Thermus
aquaticus(Thermostable)
What does PCR need?
• Template (the DNA you are exploring)
• Sequence-specific primers flanking the
target sequence , Forward & Reverse.
• Nucleotides (dATP, dCTP, dGTP,
dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer, containing salt of Taq
Polymerase
Steps of PCR
• Denaturation of template into single strand
• Annealing of primers to each original
strands for new strand synthesis
• Extension of the new strands from the
primers.
These reactions may be carried out with any
DNA polymerase and results in the
synthesis of defined portions of the original
DNA sequence.
How does PCR work?
• Heat (94oC) to denature DNA
strands
• Cool (64oC) to anneal primers
to template
• Warm (72oC) to activate Taq
Polymerase, which extends primers
and replicates DNA
• Repeat multiple cycles
 Denaturing Template
Heat causes DNA strands to separate
5’ 3’

3’ 5’
Denature DNA strands 94oC

5’ 3’

3’ 5’
 Annealing Primers
•Primers bind to the template sequence
•Taq Polymerase recognizes double-stranded substrate

5’ 3’

3’ 5’

Primers anneal 64oC

5’ 3’
5’ 3’ 3’ 5’
3’ 5’
 Taq Polymerase Extends
•Taq Polymerase extends primer
•DNA is replicated
5’ 3’
5’ 3’ 3’ 5’
3’ 5’

Extend 72oC

5’ 3’
5’ 3’ 3’ 5’
3’ 5’

Repeat denaturing, annealing, and extending 40 cycles

 The target product is
made in the third cycle
5’ 3’
3’ 5’
Cycle 1 3’
5’ 3’ 5’

3’
5’
3’ 5’
Cycle 2 5’ 3’
3’ 5’

3’ 5’
Cycle 3 5’ 3’
3’ 5’
5’ 3’
 The target sequence
• Chromosome 8
• Intron of tissue plasminogen activator (TPA) gene
• Alu-TPA25

Amplified Region

5’ Exon 8 Alu Exon 9 Exon 10 3’

Intron 8
 Alu-TPA25
• In a non-coding region of your DNA
• Not associated with any disease or disorder
• A member of Alu-repeat family
• One of 28 Alu repeats within the TPA gene
Amplified Region

5’ Exon 8 Alu Exon 9 Exon 10 3’

Intron 8
 Alu repeats
• Short (282 bp) repetitive elements flanked by direct
repeats
• They appear to be the result of retrotranspositions.
Transposable elements that resulted from reverse
transcription from RNA.
• Occur ~300,000 times in the human genome
• Named for the Alu-1 restriction site within the
element
 Evolutionary Significance
of Alu-TPA 25
• Highly conserved
• Inserted in the last 1,000,000 years
• Dimorphic (+/+, +/-, -/- )
• Used in population genetics, paternity
analysis, and forensics
 Protocol Highlights
Genomic DNA extraction
❶ Instagene = Chelex (cation exchange resin) binds
cellular MgCl2

❷ 56oC loosens connective tissue and inactivates

DNAses

❸ 100oC ruptures cell membranes and denatures

proteins
 Instagene extraction
Cell membrane

Nuclear membrane
Mg++
Genomic
DNA
Mg++ Mg++

Mg++
Heat disrupts membranes Mg++

Mg++ Instagene matrix binds

released cellular Mg++
 Protocol Highlights
✪ PCR reaction preparation
 Insure no Instagene matrix is added to reaction

Mix DNA and Master Mix thoroughly

✪ Program PCR machine and cycle

 Why is the Master Mix
ORANGE?
• Master mix contains
– reagents needed for PCR amplification
– red and yellow loading dyes
– glycerol to make samples sink

• Amplified samples can be loaded

directly onto agarose gels
 Micropipet Use
1 Twist dial to desired volume
2 Pick up pipet tip
3 Press plunger to first, soft stop
4 Insert pipet tip into solution to be
transferred
5 Slowly release plunger to retrieve liquid
6 Move pipet tip into desired tube
7 Press plunger past first stop to second, hard
stop to transfer liquid
 PCR Results….
• Alu-TPA25 is dimorphic No insertion: 660 bp
so there are two possible
PCR products:
Alu insertion: 960 bp
660 bp 330 bp each
960 bp
300 bp Alu insert
Amplified Region

5’ Exon 8 3’
Alu Exon 9 Exon 10

Intron 8
Actual Alu-PCR Results
+ - +/-

960 bp
660 bp

+ - +/-
End Of
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