Molecular Biology Lecture 6 Chapter 4 Molecular Cloning Methods Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

4-2 Lecture outline Polymerase chain reaction Technique Application Sample problems

4-3 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

4-4 Step 1Denaturation Incubate reaction mixture at 95 degrees DNA template Primers (2) Taq DNA polymerase dNTPs Buffer Reaction mixture 5’ 3’ Cycle 1 Polymerase Chain Reaction

4-5 Step 2Annealing Incubate reaction mixture at annealing temperature (5 degrees below Tm of primers) 5’ 3’ 5’ Cycle 1 Polymerase Chain Reaction

4-6 Step 3Elongation Incubate reaction mixture at 72 degrees 5’ 3’ 5’ Cycle 1 Polymerase Chain Reaction

4-7 5’ 3’ 5’ Cycle 2 Step 1Denaturation Incubate reaction mixture at 95 degrees Polymerase Chain Reaction

4-8 5’ 3’ 5’ Cycle 2 Step 2Annealing Incubate reaction mixture at annealing temperature (5 degrees below Tm of primers) 5’ Polymerase Chain Reaction

4-9 5’ 3’ 5’ Cycle 2 5’ Step 3Elongation Incubate reaction mixture at 72 degrees Polymerase Chain Reaction

4-10 5’ 3’ 5’ Step 1Denaturation Incubate reaction mixture at 95 degrees Cycle 3 Polymerase Chain Reaction

4-11 5’3’ 5’ Step 1Denaturation Incubate reaction mixture at 95 degrees Cycle 3 Polymerase Chain Reaction 5’3’ 5’

4-12 5’3’ 5’ Step 2Annealing Incubate reaction mixture at annealing temperature (5 degrees below Tm of primers) Cycle 3 Polymerase Chain Reaction

4-13 5’3’ 5’ Only this product will accumulate Step 3Elongation Incubate reaction mixture at 72 degrees REPEAT FOR A TOTAL OF 30 CYCLES Cycle 3 Polymerase Chain Reaction

4-14 AGCTTCTCGCCATTG CGCTCAATTGCGCTA TCGAAGAGCGGTAAC GCGAGTTAACGCGAT A)Design two primers to amplify this DNA fragment B)Design two primers to clone this DNA fragment in the EcoR1 site of pUC18 C) Design two primers to clone this DNA fragment in the EcoRI and HindIII sites of pUC18 EcoRI = GAATTCHindIII = AAGCTT Polymerase Chain Reaction Sample problem 1

4-15 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

4-16 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 X GTCGATCAAT 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 #25’ ___________________________________________ BamH1 = GGATCC HindIII = AAGCTT ATGAGAGGATCG GGATCCGCATGC AAGCTT RBS6Xhis ATGAGAGGATCG ACGGATCCGCATGC AAGCTT RBS6Xhis