Lab 3: Polymerase Chain Reaction (PCR) of Target DNA Fragment Introduction

In molecular biology, the polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling reactions for DNA melting, annealing and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a thermostable DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps:

Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 9498 C for 2030 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the Ta (annealing temperature) is about 3-5 degrees Celsius below the Tm (melting temperature) of the primers used. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq DNA polymerase has its optimum activity temperature at 75 80 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction.

In PCR, a pair of primer is used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 nucleotides, and they match exactly the beginning and the end of the DNA fragment to be amplified. Pairs of primers should have similar Tm, as annealing in a PCR occurs for both simultaneously. The following formula is used to calculate the Tm of DNA primer: Tm = 62.3oC + 0.41oC (% G+C) 500/length

Methodology
1. Label 0.5 ml microtube according to your group number. 2. Prepare the PCR mixture as indicated below (transfer the reagent according to the sequence, a to h): Reagent a. Distilled water b. 10X buffer c. MgCl2 (25 mM) d. dNTPs (2.5 mM) e. Forwardprimer (10 M) f. Reverse primer (10 M) g. Plasmid h.Taq DNA polymerase Total volume: Volume (l) 18 2.5 1.5 0.5 0.5 0.5 1 0.5 25 Final concentration ? ? ? ? ? -

3. Calculate the final concentration of each reagent.

4. Conduct the PCR as follows: Reaction Initial denaturation Denature Annealing Extension Final extension Temperature (oC) 94 94 55 72 72 Time 3 min 30 sec 50 sec 1 min 30 sec 7 min

5. Run the amplified DNA fragment on an agarose gel electrophoresis with an appropriate DNA marker.

Questions
1. In the conduct of PCR experiment two extra reactions, namely initial denaturation and final extension are included? Give reasons why these two reactions are used.

2. If a student use the following DNA primers in PCR. Calculate the Tm of both primers. Forward primer 5' gcc agg gcc ctt att atg tct tcc gta ttc g 3' Reverse primer 5' ata tca ata ccc cca gtc gga ggt gtc g 3'

3. Based on the X gene sequence given to you, design a forward and reverse primers that amplified the whole sequence. Calculate the Tm of both primers and suggest the appropriate Tm to be used in PCR.

Appendix I Vector Map, DNA Sequence and Primers

Forward primer,

5 GCTGATGTCGACGAGCGTAC 3

The shaded area is the sequence for Sal I endonuclease recognition site

Reverse primer = 5 GTTATCGCTTAGCCGACGCC 3

The shaded area is the sequence for Blp I endonuclease recognition site (Note: remember, this the reverse primer, intended to copy and amplify the complementary strand)

The entire DNA sequence for a X gene in pBAD vector. (The first codon is in green and underlined whereas the last codon is in red and also underlined)

AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACC AAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAA CAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCA TAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCA TACCCGTTTTTTTGGGCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATACCCATGGGCTCTG GATCCGGTGATGACGATGACAAGCTCGCCCTTATGAAAGCGATCTTAATCCCATTTTTATCTCTTCTGATT CCGTTAACCCCGCAATCTGCATTCGCTCAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAG TCGTCATGGTGTGCGTGCTCCAACCAAGGCCACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCAA CCTGGCCGGTAAAACTGGGTTGGCTGACACCGCGCGGTGGTGAGCTAATCGCCTATCTCGGACATTACCAA CGCCAGCGTCTGGTAGCCGACGGATTGCTGGCGAAAAAGGGCTGCCCGCAGTCTGGTCAGGTCGCGATTAT TGCTGATGTCGACGAGCGTACCCGTAAAACAGGCGAAGCCTTCGCCGCCGGGCTGGCACCTGACTGTGCAA TAACCGTACATACCCAGGCAGATACGTCCAGTCCCGATCCGTTATTTAATCCTCTAAAAACTGGCGTTTGC CAACTGGATAACGCGAACGTGACTGACGCGATCCTCAGCAGGGCAGGAGGGTCAATTGCTGACTTTACCGG GCATCGGCAAACGGCGTTTCGCGAACTGGAACGGGTGCTTAATTTTCCGCAATCAAACTTGTGCCTTAAAC GTGAGAAACAGGACGAAAGCTGTTCATTAACGCAGGCATTACCATCGGAACTCAAGGTGAGCGCCGACAAT GTCTCATTAACCGGTGCGGTAAGCCTCGCATCAATGCTGACGGAGATATTTCTCCTGCAACAAGCACAGGG AATGCCGGAGCCGGGGTGGGGAAGGATCACCGATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACG CGCAATTTTATTTGCTACAACGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATC AAGACAGCGTTGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTGTT TATCGCCGGACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGGACGCTTCCCGGTC AGCCGGATAACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGGCGTCGGCTAAGCGATAACAGCCAG TGGATTCAGGTTTCGCTGGTCTTCCAGACTTTACAGCAGATGCGTGATAAAACGCCGCTGTCATTAAATAC GCCGCCCGGAGAGGTGAAACTGACCCTGGCAGGATGTGAAGAGCGAAATGCGCAGGGCATGTGTTCGTTGG CAGGTTTTACGCAAATCGTGAATGAAGCACGCATACCGGCGTGCAGTTTGTAAAAGGGCGAGCTTGAAGGT AAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGT TTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGA ACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGC

CAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA ACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGG CGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTT TGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTT TCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTG TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGA TAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT GACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCAT ATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATA CCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTG GCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGC CTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAAT CTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCC CCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAG CTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAG ATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCA AACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACAT CATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGC GAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAA AAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGA AAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCT GCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTT AATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTT CCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGA AAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTA AACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACA GCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTG AGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAAT CGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAG CCATACTTTTCATACTCCCGCCATTCAGAG

Fig 1: pBAD=TOPO map (adapted from pBAD-TOPO Expression Kit manual, INVITROGEN)-