Genotyping a deletion mutation in C. elegans using PCR

Tiffany Timbers March 14, 2007

Obtaing Gene Sequence Enter gene name into

wormbase

Obtaing Gene Sequence

Click on sequence name

Obtaing Gene Sequence

Highlight and copy genomic sequence

Entering Sequence into Gentle

Gentle Workspace

Entering Sequence into Gentle Click on Magnifying glass, and paste sequence into

pop-up window

Entering Sequence into Gentle Gentle workspace after

entering a sequence

Identifying the deletion Enter strain name into

wormbase

Identifying the deletion

Click on allele

Identifying the deletion

Sequences that flank the deletion, copy these

Identifying the deletion Click on the magnifying glass, and paste in the

sequence that flanks the deletion. Click Highlight.

Do this for both the left and right flanking sequences

Identifying the deletion Highlight the sequence between the flanking sequences, and click

“EDIT”, then “Selection as new feature

This will mark the deletion in the sequence

- Design primers so that N2 amplicon is only 250-400 bp, and the amplicon of the deletion is only ~ 100bp larger - Always make sure that the wild-type amplicon is always smaller than the mutant amplicon

Primer Design Guidelines

Primer Design Guidelines

1)  Primers should be between 17-28 bp long.

2)  Should have 40-60% GC content

3)  Primer melting temperature (Tm) should ideally be between 52-58°C, but up to 64°C can work. Primer pairs should have similar melting temperatures, up to 5°C difference is allowed.

4)  Primer 3’ end should end in a G or a C, and the last 5 bases in the 3’ end of the primer should have no more than 3 G’s of C’s.

5)  Check for internal secondary structures within each primer (ex. hairpins). Generally, a 3’ end hairpin with ΔG = -2 kcal/mol or an internal hairpin with ΔG = -3 kcal/mol is OK.

6)  Check for the probability of the primer to create self dimmers. Generally, a 3’ end dimer with ΔG = -5 kcal/mol or an internal dimer with ΔG = -6 kcal/mol is OK.

Primer Design Guidelines

7) Check for the probability of the primer to create hetero- or cross dimers with other primers used in the reaction. Again, generally, a 3’ end dimer with ΔG = -5 kcal/mol or an internal dimer with ΔG = -6 kcal/mol is OK.

8) The maximum number di-nucleotide repeats (ex. atatatat) allowed is 4.

9) The maximum number of nucleotides in a run (ex. ttttttttt) allowed is 4 bp.

10) Blast each primer to ensure it will only anneal to the region you want to amplify.

You may not be able to satisfy all of these guidelines, just try to find the best ones possible and try them out…. ….if it doesn’t work try designing a new set.

Primer Design This is the region I used to

look for the F1 primer

Deletion

Testing Primers

1) Choose primers you think will work and are in the correct spot 2) Test these primers using IDTs SciTools

Make the reverse complement of your R1 primer!

Reverse Complement

Gives the sequence on the other strand

Testing Primers Enter IDT through NAPS Portal

www.michaelsmith.ubc.ca/services/NAPS/Oligonucleotide_Synthesis/

Testing Primers

1) Login (Register if new), Click on SciTools, Click on OligoAnalyzer 2) Enter Primer 3)Click Analyze

Testing Primers

Analyze

Check If: Length is between 17-28 bp GC Content is between 40-60% (with 50% being optimal)‏ Tm is between 52-58°C (Sets of primers should have similiar Tm's, up to 5°C difference is allowable)‏

Testing Primers

Click Hairpin

Then,click Submit

Testing Primers

A 3' end hairpin with a ∆G = -2 kcal/mol, or an internal hairpin with a ∆G = -3 kcal/mol is OK

Testing Primers

A 3' end homodimer with a ∆G = -5 kcal/mol, or an internal homodimer with a ∆G = -6 kcal/mol is OK

Testing Primers

Click NCBI Blast to check for primer specificity

Testing Primers

Click NCBI Blast to check for primer specificity

OK, but not optimal...c

Testing Primers

Click NCBI Blast to check for primer specificity

Just terrible, never use a primer that gives you this blast result

Testing Primers

Enter second sequence here

The same restrictions on homodimers also apply to heterodimers

Once you have found good primers...

1) Order primers through NAPS-IDT Portal 2) Test these primers on N2 initially to identify

optimal PCR conditions 3) Need to genotypt 6 N2 and 6 mutants to verify

genotype