Targeted PCR and Sanger Resequencing of Mutations

Page in need of additional improvements.

Primer Design

Sequence immediately 5' of the sequencing primer will always be undetermined, it is bested to give at least 85-100bp of space before getting to a region of importance, and sequencing will deterierorate at some level over the next ~900bp. Several different options for designing primers exist, the following information applies regardless of what option is used: Select appropriate coordinates for each primer and PCR product size based on the following:

  1. For point mutations and small indels: Design two primers ~200-350 bp upstream and ~200-350 bp downstream of each mutation to amplify a 400-700 bp fragment. Sanger sequence from only one end. One reaction per template.
  2. For IS-insertions: Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify product. Do control reactions with REL606 to verify the expected size change. Sequence from both sides for IS-insertions so that we can determine the new IS orientation and new junction sequences.
  3. For large deletions (>4kb): Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify a product for the deletion. If the product for the non-deleted ancestral genome is too large to reliably amplify (> 5 kb), then order an additional primer within the deleted interval that can amplify a 400-700 bp fragment with the original forward primer. Always do control reactions with REL606 to verify that a band appears in it for just one primer pair (the non-deleted specific pair) and that a band appears for the evolved genomes with just one pair (the deletion specific pair). Sequence the deletion from just one end. Be sure to design primer pairs such that the products of each PCR differ by >200 bp in length, so that you can tell them apart on the gel!
  4. For rearrangements, amplifications, and inversions Design primers ~200-350 bp upstream and downstream of new junctions. PCR across the junction and Sanger sequence to verify the exact junction site. Be careful to NOT design primers in repetitive regions (such as IS elements, tRNA genes, rRNA genes, etc.) that could give confusing amplification products!

Primers sequences and stocks already exist for some genes: topA

NCBI primer design

Use the NCBI primer-blast tool to design primers: http://www.ncbi.nlm.nih.gov/tools/primer-blast/

If working with REL606 or other LTEE derivatives:

  1. In the "PCR Template" box, enter "NC_012967" as the accession to search.
  2. Under "organism" in the "Primer Pair Specificity Checking Parameters" section, enter "Escherichia coli (taxid:562)".

Default settings for most other parameters are typically fine (pay attention to PCR product size).

Benchling primer design

To be entered.

PCR reaction

Perform standard PCR reactions.

In all PCR reactions use ~1 ng/ul of genomic DNA sample as template. Always use high-fidelity polymerase. Possibly "long-template" polymerase if the amplified fragments are very large (>5 kb).

Check PCR and Quantify DNA Yields

Run 2-5 l of sample + 2-5 l of glycerol load buffer on a 0.8-2% agarose gel.

Load 5 l of 0.1g/ml DNA ladder. Use either 100-bp or 1-kb DNA ladder size as appropriate.

Ladders commonly used in lab.

Link Company Description Cat # Unit Price
external New England Biolabs 100-bp ladder N3231L 500 gel lanes $212

Estimate band concentrations by eye or by saving the image in TIFF format and finding band densities with ImageJ. Compare to a reference band of similar density and determine the concentration of the original sample.

DNA purification

Previous cost analysis has revealed that it is not more expensive for the core to do the PCR purification themselves, so select PCR cleanup and sequencing.

Sequencing Reaction

UT Sanger Sequencing Page

Specific info needed here.

Analysis of sequencing results

Benchling

To be implemented

 Barrick Lab  >  ProtocolList  >  ProceduresTargetedSequencing

Topic revision: r7 - 23 Feb 2017 - 21:30:38 - Main.DanielDeatherage
 
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