PCR Worksheet. Components: 1. Thermocycler 2. Template 3. Primers (Forward and Reverse) 4. dntps 5. Polymerase. PCR Step 1: Denature

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1 Components: 1. Thermocycler 2. Template 3. Primers (Forward and Reverse) 4. dntps 5. Polymerase PCR Worksheet The DNA fragment below shows one portion of a chromosome. Items of note: 1. DNA is double-stranded (shown below: Top strand and bottom strand) 2. Strands have direction 5 to 3. DNA is ALWAYS made in this direction 3. The two strands are antiparallel (shown by the arrows, but typically just by the 5 at the beginning and 3 at the end. Usually the top strand is going left to right) 4. Individual nucleotides (specifically deoxynucleotide monophosphates or dnmps) in a strand are held together by strong covalent bonds. (left to right) 5. The strands (top to bottom) are held together by the weak hydrogen bonds of base pairing ( G:C & A:T ) 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 ::::::::::::::::::::::::::::::::::::::::::::::::: Supposing the highlighted (dotted box) region corresponds to your gene interest. Using PCR we can amplify this region. (note: the above piece of DNA is only some of a much larger piece, for example a chromosome) PCR Step 1: Denature Thermocycler raises temp to 94 C 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 1

2 Step 2: Anneal (Join together) Thermocycler lowers temp to something low, typically ~58 C This allows hydrogen bonds between base pairs to form. This means the top and bottom strands can come together (renature, anneal). However, some primers will anneal instead. This is explained by the fact that there is an overwhelming abundance of primers added and it is thought that, because primers are small in comparison, they move faster due to smaller size, and can therefore more easily sneak in and anneal. Primers are artificially synthesized and a easily commercially available. Typically they are about nucleotides (just long enough so that they only have one target). For convenience we will use fictional ones that are about 6 nucleotides long. Below the Forward and Reverse Primers are shown. Note the conditions for the primer - Is complementary (base pairs) to the target - Brackets the back of the target region - It is pointed (5 to 3 ) inward and therefore: the forward primer must anneal to the bottom strand the reverse primer must anneal to the top strand 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 :::::::::: 3 -ACGGGT-5 Reverse primer Forward primer 5 -CGGGTT :::::::::: Step 3: Extend Thermocycler raises temp to 72 C DNA Polymerase extends existing strands acc - Using free dntps - according to base pairing of free nucleotides base pairing to template Below the primers from the previous step have been extended, and the resulting product from the first cycle is shown. There are usually multiple enzymes, each polymerizing different (forward and reverse primer) strands 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 2

3 PCR Worksheet Given the following piece of DNA 5 -TCCCGGGATGCCCAAAATTCTCGAGCTACCTGCGGGTTGACTGCTACCT-3 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 3 -AGGGCCCTACGGGTTTTAAGAGCTCGATGGACGCCCAACTGACGATGGA-5 Question 1: Below, Draw the Forward and Reverse Primers annealed to the template. Denature - 94 C Anneal - 58 C 5 -TCCCGGGATGCCCAAAATTCTCGAGCTACCTGCGGGTTGACTGCTACCT-3 3 -AGGGCCCTACGGGTTTTAAGAGCTCGATGGACGCCCAACTGACGATGGA-5 Question 2: What is the substrate for the reaction? Primer is one, what is the other? Question 3: Below, after the extension, draw the resulting new strands. Anneal - 58 C 5 -TCCCGGGATGCCCAAAATTCTCGAGCTACCTGCGGGTTGACTGCTACCT-3 3 -AGGGCCCTACGGGTTTTAAGAGCTCGATGGACGCCCAACTGACGATGGA-5

4 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 5 -CGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG For convenience, instead of writing out the sequences, we will use lines to represent our strands and BOLD arrows to represent our primers. Your completed step 3 is drawn below. Notice, how there are now 2 double-stranded pieces of DNA instead of one. You have just doubled your DNA and you have two sets of template DNA, not one. Typically a PCR is repeated 30 times. Using lines and BOLD ARROWS for primer. Complete 2 more rounds. For Round 2 the denatured strands are show below. Add in bold arrows for primers in the anneal step, and draw the results after extension. Round 2 94 C Equivalent to: 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 5 -CGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG 3

5 Round 2 5 -CTACCTGCGGGTTGACTGCTACCTTCCCGGGATGCCCAAAATTCTCGAG-3 3 -GCCCAACTGACGATGGAAGGGCCCTACGGGT-5 Round 3 Question 4: Draw the results of round 3 below. Using arrows only is ok.

6 Question 5: For the target sequence below, located in chromosome 16 what are the forward and reverse primers needed (give the sequence)? How long is the FINAL PCR product? In other words, how many nucleotides long is it? Note: for educational purposes, a fictional sequence is used. The underlined portion represents PV92. It is sometimes present on chromosome 16. Other than the underlined portion, it is identical to the above DNA. Question 6: What are the primers needed to amplify the target region? Question 7: How does it compare to the above? Question 8: How long is the final PCR product? Because humans have 2 of every chromosome, it is possible to have every combination of these two DNAs. How long are the 2 copies with NO PV92 final PCR products? 2 copies WITH PV92 1 copy with, 1 copy without Affectively each different template becomes a separate reaction with a separate product. Note: For this exercise, use the DNA given above to calculate the PCR product length. However, the actual length of the PCR product is 416 nucleotides (nt) without PV92, and 731 nt with PV92. 4