Question

1. How is complimentary DNA (cDNA) made from total RNA extract? What type of enzyme and primer is needed? What are the advantages of cloning a gene from its cDNA as opposed to the genomic DNA sequence (introns)?

2. If a PCR reaction replicates both strands of the DNA each cycle, this produces exponential amplification of the product. If only one strand is replicated (as in a Sanger sequencing PCR reaction) there is a linear growth in the product. Why is this?

3. Quantitative PCR (qPCR) was described. How can this be used to quantify the amount of an mRNA in a sample? What is the template? How does SYBR green dye allow for quantification of the amount of product each cycle? How would you interpret the results on slide 17 panel A? If sample #1 takes 20 cycles to produce fluorescence intensity equal to the Ct, and sample #2 takes 21 cycles, how much more mRNA detected in sample #1 than #2?

Answer 1

cDNA(Complimentary DNA) is synthesized from mRNA by using the special enzyme Reverse transcriptase. mRNA is used as a template and reverse transcriptase synthesizes a single attended DNA molecule which in turn can be used as template for Double stranded DNA synthesis.

An oligo(dT) primer is used for the synthesis. Oligo (dT) is a sequence of deoxythymine which is used as primer for reactions catalysed by Reverse transcriptase.

As mRNA is devoid of non coding sequences (introns), the cDNA formed from it has only the coding DNA sequences as opposed to the genomic DNA which has both coding and non coding sequence. This specificity of cDNA is advantageous in translating cDNA into functional protein in a bacterial system (plasmid).