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Molecular Biology
Lecture 6
Chapter 4
Molecular Cloning Methods
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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Lecture outline
Polymerase chain reaction
• Technique
• Application
• Sample problems
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Polymerase Chain Reaction
• Objective is to produce a specific DNA sequence in-vitro
• Amplification of the target DNA can be done from minute amount of starting material
• DNA synthesis is catalyzed by a thermo stable DNA polymerase
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Step 1 Denaturation
Incubate reaction mixture at 95 degrees
DNA templatePrimers (2)Taq DNA polymerasedNTPsBuffer
Reaction mixture
5’
5’
5’
5’3’
3’
3’
3’
Cycle 1
Polymerase Chain Reaction
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Step 2 Annealing
Incubate reaction mixture at annealing temperature(5 degrees below Tm of primers)
5’
5’
3’
3’5’
5’
Cycle 1
Polymerase Chain Reaction
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Step 3 Elongation
Incubate reaction mixture at 72 degrees
5’
5’
3’
3’5’
5’
Cycle 1
Polymerase Chain Reaction
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5’
5’
3’
3’
5’
5’
Cycle 2
Step 1 Denaturation
Incubate reaction mixture at 95 degrees
Polymerase Chain Reaction
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5’
5’
3’
3’
5’
5’
Cycle 2
Step 2 Annealing Incubate reaction mixture at annealing temperature (5 degrees below Tm of primers)
5’
5’
5’
5’
Polymerase Chain Reaction
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5’
5’
3’
3’
5’
5’
Cycle 2
5’
5’
5’
5’
Step 3 ElongationIncubate reaction mixture at 72 degrees
Polymerase Chain Reaction
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5’
5’
3’
3’
5’
5’
5’
5’
5’
5’
Step 1 DenaturationIncubate reaction mixture at 95 degrees
Cycle 3
Polymerase Chain Reaction
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5’ 3’
5’5’
5’
Step 1 DenaturationIncubate reaction mixture at 95 degrees
Cycle 3
Polymerase Chain Reaction
5’ 3’
5’
5’
5’
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5’ 3’
5’
5’
5’
Step 2 AnnealingIncubate reaction mixture at annealing temperature (5 degrees below Tm of primers)
Cycle 3
Polymerase Chain Reaction
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5’ 3’
5’
5’
5’
Only this product will accumulate
Step 3 ElongationIncubate reaction mixture at 72 degrees
REPEAT FOR A TOTAL OF 30 CYCLES
Cycle 3
Polymerase Chain Reaction
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AGCTTCTCGCCATTG CGCTCAATTGCGCTATCGAAGAGCGGTAAC GCGAGTTAACGCGAT
A) Design two primers to amplify this DNA fragment
B) Design two primers to clone this DNA fragmentin the EcoR1 site of pUC18
C) Design two primers to clone this DNA fragment in the EcoRI and HindIII sites of pUC18
EcoRI = GAATTC HindIII = AAGCTT
Polymerase Chain Reaction
Sample problem 1
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RT-PCR Can be used for cloning
• Restriction enzyme sites can be added to the cDNA of interest
• Able to generate sticky ends for ligation into vector of choice
• 2 sticky ends permits directional cloning
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6) (6 points) For your independent research project, your supervisor is asking you to clone the coding region of gene X (see below) in the bacterial expression vector pQE30 using the polymerase chain reaction (PCR). Write the sequence of two oligonucleotides that will allow you to clone the coding sequence in the vector. The recombinant protein must be as short as possible. Note: The coding sequence must be in frame with the ATG of the vector. The start and stop codons of gene X are underlined.Coding sequence of gene XGTCGATCAAT ATGGAACATG TTTACTCCAA ACCACCGCAC ACCAATTATG GAAACCAAGC CGGAAAAGAA TTCCGGTGGA GAGCGAAAAA AAAGGATTCC GAATCGTGAA CTGCCAAAAA CATTTTGAAG CCAACGATTC CGACGTCATC CTCGCCACCC TAGCTAAATC AGGCACCACT TGGTTAAAAG CTCTTCTCTT TGCTCTCATT CACCGACACA AGTTCCCAGT TTCTGGCAAG CATCCTCTTC TGAAACAGCA GTAGCAGCGT TTAAAGGGAA GTTTATT Oligo #1 5’ ___________________________________________Oligo #2 5’ ___________________________________________BamH1 = GGATCCHindIII = AAGCTT
ATGAGAGGATCG GGATCCGCATGC---------AAGCTTRBS 6Xhis
ATGAGAGGATCG ACGGATCCGCATGC---------AAGCTTRBS 6Xhis
