PCR Gene.

Order Discription. 1. Why are two primers required to amplify a gene by PCR? (10 marks) A primer in PCR reaction is known as a single stranded DNA sequence. However, as a primer will only define a specific region within a DNA that need to be copied, therefore the two primers is required in order to a gene amplification as each primer will bind complementary to a different DNA strand. This makes the binding of primer to either side of the strands of target DNA is more likely. By another word, each primer will recognize and bind to one strand of the double stranded DNA, and these strands form the specific region of the target DNA sequence that will be amplified. Primer allows DNA polymerase to bind and start replicating DNA. 7/10 almost there but could be expressed more clearly 2 reasons (5 marks each): 1. To define both of the outer limits (start and end) of the sequence being amplified. 2. Because one primer will only amplify a single strand of the template DNA. To amplify dsDNA template, one primer is needed for each of the two DNA strands. 2. What is the reverse complement of this sequence? (written 5’-3’) 5’-GCT ATT TCG CAG TCC AGC (5 marks) The reverse complement of the above sequence is: 5’ GCT GGA CTG CGA AAT AGC 3’ 5/5 3. What is the “GC clamp” and why is this an important feature of a PCR primer? (10 marks) The GC clamp is known as the G’s or C’s bases that present at the 3’ end of a primer, they are located within the last five bases of primers. Yes Regarding to the known strong bonding of both bases (G and C), this GC clamp got its importance as at the 3’ end of a primer these G’s and C’s bases give strong specific binding. However, it should be in account that more than 3 G’s or C’s might affect the primer binding specificity so avoid that. 5/10 G-C interaction (3 hydrogen bonds) is stronger than A-T interaction (2 hydrogen bonds). GC clamp prevents “breathing” (3’ end of the primer dissociating from the template DNA). Tight binding of primer to template at this end is important for efficient binding of the DNA polymerase, which will replicate the DNA in a 5’ to 3’ direction. 4. Which of the following primers will form a hairpin structure, and why might this make the primer unsuitable for PCR? Primer A: 5’-CGC TGA CGT AAT GCT AGC CAT Primer B: 5’-CTT ATG GCT GTA CGT CTA CTG Primer C: 5’-CGC TGA CGT ATA CGT CAG CGG (15 marks) Primer C: 5’-CGC TGA CGT ATA CGT CAG CGG is more likely to form hairpin structure. The hairpin structure has direct relation to the amplification and that by affecting the primer designing. Thus, the amplification efficiency is decreased and that happened by forming complementarity at the 3’ end of the primer, subsequently this leads to amplifying of primer themselves due to the increasing of spurious products possibility (Singh V, et al. 2000). Not quite… You have correctly identified primer C as the hairpin-forming primer. This makes it unsuitable for PCR because it binds strongly to itself, making those bases unavailable for binding to the template DNA. This will reduce the yield of PCR product. 7/15 5. What is the ideal melting temperature for a PCR primer, and why? (20 marks) Use Oligo Analyzer https://www.idtdna.com/calc/analyzer to calculate the melting temperatures of primers A to C above. (10 marks) The ideal melting temperature for PCR primer is controversially different from one source to another. According to the Oligo Analyzer website the ideal melting temperature is 60–64°C. However, as far as I found 55-65°C is considered the best melting temperature for PCR primer in most applications, with the ideal is 60°C. In order to produce a good PCR result the melting temperature of primers should be within the ideal range. There are different ways to calculate the melting temperature but one is considered the best and accurately way to use, which is the nearest neighbour thermodynamic calculation. However, when high temperature is used, the primer with lower melting temperature will not appropriately function why not?, as well as a primer with too high melting temperature such as 70 °C can lead to secondary annealing what is this?, and that affect the result (Dieffenbach C, et al. 1993). Indeed, the difference between the melting temperature of two primers should not be exceeds 5 °C good as this will decrease the amplification efficiency and also affects binding specificity of primers. Also, is important to mention that the melting temperature depends on how many bases the primer has and the % of G and C bases on the primer too. Good. Tm should match that of other primer, and be around 50-60 C, and about 5 C higher than the annealing temperature used in the PCR cycler. Too low => non specific products may form, too high => there may be low yield of PCR product. Melting temperatures: Primer A: 57.5 ºC Primer B: 52.3 ºC Primer C: 60 ºC all correct 18/30 6. I have designed a PCR primer to detect my gene of interest (cagA) in Helicobacter pylori. I need to be sure that the PCR will only bind to the H. pylori cagA gene and not to genes from the human host or other bacteria. How can I find out if the primer is specific to H. pylori cagA? Carry out this check for me, and describe both the method you used, and your results. Hp_cagAF: 5’- TTCATGGGCGTGTTTGATG (30 marks) Method: The NCBI website was used to blast the primer sequence, (BLAST nucleotide database). First of all, NCBI page was opened, then BLAST was chosen from that page and finally nucleotide BLAST was chosen and the primer sequence was blasted. However, when the primer was blasted, all results that came up were 100% identity to the gene of interest (cagA) in Helicobacter pylori. This indicates that this primer is unique to the (cagA) gene and will not bind to human genes or other bacterial genes, thus will be mainly bind to H. pylori cagA gene. 17/30 a good start but more detailed analysis needed for full marks. Also showing your results and providing more information such as % similarity would have been good here. BLAST searching for the primer sequence against human genome will give some 100% matches, suggesting that the primer could also amplify contaminating human genes. TOTAL = 59/100 References: Dieffenbach C, et al. (1993). General concepts for PCR primer design. Genome Research; (3); 830-837 Singh V, et al. (2000). General concepts for PCR primer design. Molecular Biology Today; 1(3); 67- 69