project i verifying the restriction map of a dna insert
TRANSCRIPT
Project IProject I
Verifying the restriction Verifying the restriction map of a DNA insertmap of a DNA insert
ObjectivesObjectives
Find out which gene you have Find out which gene you have (Bioinfo)(Bioinfo)
Generate a theoretical map Generate a theoretical map (Bioinfo)(Bioinfo)
Verify map experimentallyVerify map experimentally Determine orientationDetermine orientation
X Delimits Right & LeftX Delimits Right & Left
Ex. If Pst is the insertion site: Bam is to the left.If Xba is the insertion site, then Bam is to the right
Determining Relative Determining Relative OrientationOrientation
X XA A
X XA A
Orientation 1
Orientation 2
ObjectivesObjectives
Use PCR to amplify and Use PCR to amplify and mutagenize the LacZ gene in mutagenize the LacZ gene in pUC19pUC19
Use PCR to add appropriate Use PCR to add appropriate restriction sites to the ends of the restriction sites to the ends of the LacZ gene to allow cloningLacZ gene to allow cloning
DNA Replication DNA Replication & Amplification& Amplification
The Polymerase Chain The Polymerase Chain ReactionReaction
PolymerasesPolymerases
5’…GTACT3’…CATGAATGCTGCATTTGCGGGCATTACTC…5’
Polymerase
Primer
-OH
3’OH end
TACGACGTAAACGCCCGTAATGAG
DNA or RNATemplate
2 Types of DNA Polymerases :2 Types of DNA Polymerases : DNA dependentDNA dependent :
Requires a DNA template Synthesize DNA
Ex. Taq polymeraseRNA dependent
Requires an RNA template Synthesize DNA (cDNA)
Ex. Reverse transcriptase
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The Polymerase Chain Reaction-The Polymerase Chain Reaction-PCRPCR
Repetitive replication of a given region Repetitive replication of a given region of DNAof DNA
Allows the Allows the exponentialexponential amplification of a amplification of a given region of DNAgiven region of DNA
Increases the relative representation of Increases the relative representation of the region of interestthe region of interest
Allows the isolation of a given region of Allows the isolation of a given region of DNADNA
PCR-1PCR-1stst Cycle Cycle
5’5’3’3’
3’
3’
5’
5’
Denaturation (95Denaturation (95ooC)C)
Annealing of primers (Tm)Annealing of primers (Tm)
5’CATACCGTGGGGTGCA………..ACGCGTTGCGATGGCA3’
3’GTATGGCACCCCACGA………..TGCGCAACGCTACCGT5’5’CCGTGGGGT3’>
<3’GGAACGGTACCGT5’
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Extension (72Extension (72ooC)C)
3’
3’
5’
5’
5’CATACCGTGGGGTGCA………..ACGCGTTGCGATGGCA3’
3’GTATGGCACCCCACGA………..TGCGCAACGCTACCGT5’5’CCGTGGGGT3’>
<3’GGAACGGTACCGT5’
PCR-2PCR-2ndnd Cycle Cycle3’5’
3’ 5’
---------------------------------- --------------------------------------5’ 3’5’3’
AnnealingAnnealing
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3’
3’
3’5’
3’ 5’
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--------------------------------------5’
5’
DDenaturationenaturation
--------------- /Extension/Extension
PCR-Subsequent CyclesPCR-Subsequent Cycles--------------------
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OnlyOnly this template is amplified this template is amplified
exponentially: exponentially: 22nn times times------------------------ ------------------------
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------------------------ ------------------------------------------------ ------------------------32 times total32 times total
Review of PCR CyclesReview of PCR Cycles
PCR Primers:PCR Primers: Short single stranded nucleotide sequences Short single stranded nucleotide sequences
complementary to the targetscomplementary to the targets 15-30 nucleotides15-30 nucleotides Used in excess as compared to target to Used in excess as compared to target to
favor primer annealing rather than template favor primer annealing rather than template self annealingself annealing
Review of PCR CyclesReview of PCR Cycles
Annealing:Annealing: Temperature at which primers anneal to Temperature at which primers anneal to
complementary target sequencescomplementary target sequences Must be below primer TmMust be below primer Tm Must be a temperature that allows both Must be a temperature that allows both
primers to annealprimers to anneal Usually between 55-75Usually between 55-75ooCC
Review of PCR CyclesReview of PCR Cycles
Extension:Extension: Carried out at temperature optimum for Carried out at temperature optimum for
DNA polymeraseDNA polymerase Usually 72-75Usually 72-75ooC for Taq polymeraseC for Taq polymerase
Taq PolymeraseTaq Polymerase Isolated from a thermophillic bacterium Isolated from a thermophillic bacterium
Thermus aquaticusThermus aquaticus Stable at the high temperatures (~95Stable at the high temperatures (~95ooC) C)
used for denaturing DNAused for denaturing DNA No exonuclease activityNo exonuclease activity
No proof readingNo proof reading Has deoxynucleotidyl transferase activityHas deoxynucleotidyl transferase activity
Template independent polymerase activityTemplate independent polymerase activity Adds dA to free 3’OH endsAdds dA to free 3’OH ends
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PrimersPrimers Characteristics:Characteristics:
Short oligonucleotides complementary to Short oligonucleotides complementary to sequences that flank the region of interestsequences that flank the region of interest
Establish the point of initiation of replicationEstablish the point of initiation of replication
Establish the point of termination of Establish the point of termination of replicationreplication
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Primer DesignPrimer Design
Autocomplementarity:Autocomplementarity:
5’GGGGCCCC3’
GGGG
CCCC
Complementarity of the Pair:Complementarity of the Pair:
5’GGGGAAAA3’
3’CCCC TTTT5’
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Primer DesignPrimer Design
5’ complementarity5’ complementarity
3’…………….ATGGGTATTGGCC…………………..-5’Template
CCATAACCGG-OH3’ 5’CGA
3’ complementarity3’ complementarity
3’…………….ATGGGTATTGGCC…………………..-5’Template
TACCCATAACC TA-OH3’
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Primer DesignPrimer Design
5’
5’3’
3’
Region of interest
Region of interest3’
3’
3’
3’
Correct orientationWrong orientation
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ProblemProblem
You wish to amplify the sequence You wish to amplify the sequence represented by the box. Which represented by the box. Which primer pair represent the correct primer pair represent the correct orientation to accomplish this?orientation to accomplish this?
5’-AAAAAAAAAAAA GGGGGGGGGGGGG-3’
1- AAAAAA2- TTTTTT3- GGGGGG4- CCCCCC
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Utility of PCRUtility of PCR
Amplification and isolation of a given region Amplification and isolation of a given region by changing its relative representationby changing its relative representation Between 100bp and 10KpbBetween 100bp and 10Kpb
Screening to determine the presence of a Screening to determine the presence of a sequence of interestsequence of interest Presence or absence of an amplification productPresence or absence of an amplification product
Site directed mutagenesisSite directed mutagenesis Used to add or remove nucleotides from the Used to add or remove nucleotides from the
original templateoriginal template