Question

Instructions: Design primers for genotyping a 200bp deletion in gene Y6B3B.10 (WIT 7470 bp). Copy and Paste in the space below deletion in gene Lng-1gk327) Q1. (2 pts) Annotate (highlight) your primers. Provide your sequences below in the provided space as needed for ordering. orward Q2. (I pt) In a single sentence explain what the functional group must be on your primer and what is its purpose? the 3' end of Q3.(1 pt) List 4 key properties that your primers are designed with

Answer 1

Q.1)

Forward sequence - 5'-t c c a c t g t t c a t g g t c a c c -3'

Reverse sequence - 5'-g a a a t c t g a a a t t t t t a t t c -3'

1. The primer at the 3' end should not have more than 3 Cytosine/Guanine bases in the last 5 bases.

​​​​​​​Q.2) The 3'-end has Hydroxyl group that is involved in attachment of nucleotides as a part of polymerization reaction.

Q.3)   1) They have size in the optimal range that is 17-28 bp.

2) Melting temperature is below 64°C. ( Can specify from wormbase )

3) Primer ends in a G/C.

4) Maximum number of nucleotides in a run is less than 4bp. (As seen in the above sequence)

Note:- Amplicons should range from 250-400bp depending upon the size of deletion that is 200bp. NCBI blast may prove effective in aligning the primer sequence according to the deletion region. Primer testing and Reverse complement needs to be performed.