Question

1.) How many kinds of reverse transcriptases are there ? 2.) What would normally be a proper order to assemble yout RT reaction mix ? Your PCR mix ? Why is the order important ? 3.) Why do you need many PCR cycles to see reaction products ? 4.) Why are multiple product bands sometimes seen during PCR reactions ? How can this problem be minimized ? 5.) Betaine is often included in transcription reactions. What is betaine ? What is its function in the reaction ? 6.) What is the purpose of a pilot PCR experiment ? 7.) Many PCR primers contain 2-4 C's and/or Gs at the 3' end, a so-called GC clamp. What is the reason for including these specific bases at the 3' end of a primer ? 8.) How may the length of a PCR primer affect the resulting product formation ? Consider primers that are longer and shorter than an "optimal" primer length.

Answer 1

1.Reverse transcriptase is an RNA dependent DNA polymerase that catalyses the conversion of RNA template molecules into a DNA double helix and are commonly used to produce cDNA libraries.Reverse transcriptases are of three kinds.

i)RNA dependent DNA polymerase

ii)RNase H and,

iii)DNA dependent DNA polymerase.

3.Many PCR cycles are used to amplify the target DNA.The number of cycles is usually carried out 25-35 times but may vary upon the amount of DNA input and the desired yield of PCR product.

5. Betaine is a 5 molar PCR reagent .Its formula is (CH3)₃N⁺CH₂COO^-.Betaine improves the co amplification  of two alternatively spliced variants of the prostate specific membrane antigen mRNA as well as thetamplification of coding cDNA region of c-jun and is generally applicable to ameliorate the amplification of GC-rich DNA sequences.

7.The presence of C and G bases with in the last five bases in the 3' end of a primer helps promote specific binding at the 3' end due to the stronger bonding of G and C bases.

8.The optimal length of PCR primers is generally 18-22 base pairs.The length is good enough for adequate specificity and short enough for primers to bind easily the template at the annealing temperature. As the length of primer increases,the times to find the particular sequence in the human genome decreases.Primer sequences need to be choosen to uniquely select for a region of DNA avoiding the possibility of mishybridisation to a similar sequence near by and the efficiency of annealing decrease with increased primer length.