Project I Verifying the restriction map of a DNA insert.

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

Mapping of Unk. PlasmidsMapping of Unk. Plasmids

Vector pUC19Vector pUC19

Cloning in pUC19Cloning in pUC19

X

MCS Digested with X

Cloning in pUC19Cloning in pUC19

X

X

+Insert

Determining Insertion SiteDetermining Insertion Site

Cut with XX

X

+Insert

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

Project IIProject II

Site directed Site directed mutagenesis of LacZmutagenesis of LacZ

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

--------------

----------------------------------

-------------------------------

3’

3’

3’5’

3’ 5’

----------------------------------

--------------------------------------5’

5’

DDenaturationenaturation

--------------- /Extension/Extension

PCR-Subsequent CyclesPCR-Subsequent Cycles--------------------

-------------------

OnlyOnly this template is amplified this template is amplified

exponentially: exponentially: 22nn times times------------------------ ------------------------

------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------

------------------------ ------------------------------------------------ ------------------------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