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Pcr

Pcr

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03/18/2014

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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’
3’ 5’

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’

Cycle 1 Cycle 2
3’

3’

5’

3’

3’

5’

3’ 5’ 3’

5’ 3’ 5’

5’ 3’ 5’

Cycle 3

3’ 5’ 3’ 5’

5’ 3’ 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 Intron 8 Exon 9 Exon 10 3’

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
‚ Instagene = Chelex (cation exchange resin) binds

cellular MgCl2 ƒ 56oC loosens connective tissue and inactivates DNAses „ 100oC ruptures cell membranes and denatures proteins

Instagene extraction
Cell membrane Nuclear membrane

Genomic DNA
Mg++

Mg++

Mg++

Heat disrupts membranes
Mg++

Mg

++

Mg++

Instagene matrix binds released cellular Mg++

Protocol Highlights
7 PCR

reaction preparation

Insure no Instagene matrix is added to reaction  Mix DNA and Master Mix thoroughly
7 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 2 3 4 Twist dial to desired volume Pick up pipet tip Press plunger to first, soft stop 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 so there are two possible PCR products: 660 bp 960 bp
Amplified Region
5’

No insertion: 660 bp

Alu insertion: 960 bp 330 bp each

300 bp Alu insert
Exon 8

Alu
Intron 8

Exon 9

Exon 10

3’

Actual Alu-PCR Results
+ - +/-

960 bp 660 bp

+ - +/-

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