Part 1) A researcher planes to amplify the CDS of the SAM1 gene. The partial seq

Question

Part 1) A researcher planes to amplify the CDS of the SAM1 gene. The partial sequence below shows the coding sequences for the N-terminus and C-terminus of Sam1p. Design two 18-nucleotide long priers to amplify the sequence the SAM1 CDS.

5'- ATGGCCGGTACATTTTTATTC........(CDS).........TCCAAGACTTTGAAGTTCTAA- 3'

Part 2) Most of the primers that we are using for the PCR have Tms that are slightly lower than the annelaing temperature used in the PCR reactions. Thus, less than half of the target sites are expected to annel with the primers in each cycle. Why would investigators choose to design primers that will not be fully annealed with the DNA template?

Explanation / Answer

1. Forward primer: 5'ATG GCC GGT ACA TTT TTA3'

Reverse primer: 5' TTA GAA CTT CAA AGT CTT3'

2. If Tm is too low to primer annealing temperature the non-specific nucleotide pairing will take place frequently throughout DNA template.

If Tm is higher than primer annealing temperature, either specific or non-specific nucleotide pairing with template DNA does not occur.

So investigators always prefer the stringent temperature (Tm +/- 2oC) for primer annealing with DNA template that would not support mispairing and/or complete pairing with DNA templates in a single PCR cycle.

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