Pcr
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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’
3’ 5’
3’
3’
5’
5’
Taq Polymerase Extends •Taq Polymerase extends primer •DNA is replicated 3’
5’ 3’
5’
3’
3’
5’
5’
Extend 72oC 5’ 3’
3’ 5’
3’
3’
5’
5’
Repeat denaturing, annealing, and extending 40 cycles
The target product is made in the third cycle 5’
Cycle 1
5’
3’
Cycle 2
5’
3’
5’ 3’
5’ 3’
5’
5’
3’ 5’
3’
Cycle 3
3’
3’
3’ 5’
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
Intron 8
Exon 9
Exon 10
3’
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 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++
Heat disrupts membranes Mg++
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 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
No insertion: 660 bp
Alu insertion: 960 bp 330 bp each
300 bp Alu insert Amplified Region 5’
Exon 8
Alu Intron 8
Exon 9
Exon 10
3’
Actual Alu-PCR Results + - +/-
960 bp 660 bp
+ - +/-
End Of Show
(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’
3’ 5’
3’
3’
5’
5’
Taq Polymerase Extends •Taq Polymerase extends primer •DNA is replicated 3’
5’ 3’
5’
3’
3’
5’
5’
Extend 72oC 5’ 3’
3’ 5’
3’
3’
5’
5’
Repeat denaturing, annealing, and extending 40 cycles
The target product is made in the third cycle 5’
Cycle 1
5’
3’
Cycle 2
5’
3’
5’ 3’
5’ 3’
5’
5’
3’ 5’
3’
Cycle 3
3’
3’
3’ 5’
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
Intron 8
Exon 9
Exon 10
3’
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 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++
Heat disrupts membranes Mg++
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 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
No insertion: 660 bp
Alu insertion: 960 bp 330 bp each
300 bp Alu insert Amplified Region 5’
Exon 8
Alu Intron 8
Exon 9
Exon 10
3’
Actual Alu-PCR Results + - +/-
960 bp 660 bp
+ - +/-
End Of Show
