Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

Abstract

Source: Tsujimoto, H. et al., Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti. J. Vis. Exp. (2021).

This video demonstrates the gradient polymerase chain reaction for optimization of the annealing temperature of primers, which sets gradient annealing temperature along wells. The effect of different annealing temperatures is determined by the yield of amplified products that helps to optimize the reaction condition.

Protocol

1. Scanning for single nucleotide polymorphisms (SNPs), HRMA primer design, and primer validation SNP identification in wild-type laboratory colony mosquitoes Select the target exon to disrupt proper polypeptide translation. NOTE: The target should be close to the start codon or amongst key residues required for protein function. The shorter the exon (e.g., ≤200 bases), the more difficult it is to target and analyze. Avoid editing close to the boundaries of an exon, …

Representative Results

Figure 1: SNP identification. Schematic representation of multiple sequence alignment of AaeZIP11 fragment from wild-type. In red are the SNPs, and in green are the fragments free of SNPs; this SNP-free region is suggested for sgRNA and primer design. Abbreviations: SNP = single nucleotide polymorphism; sgRNA = single guide RNA; LVP = Liverpool strain.

Materials

70% Ethanol			70% ethanol solution in water
96-well PCR and Real-time PCR plates	VWR	82006-636	For obtaining genomic DNA (from the mosquito leg)
96-well plate templates			House-made printed, for genotype recording
Bio Rad CFX96	Bio Rad		PCR machine with gradient and HRMA capabilities
Diversified Biotech reagent reservoirs	VWR	490006-896
Exo-CIP Rapid PCR Cleanup Kit	New England Biolabs	E1050S
Glass Petri Dish	VWR	89001-246	150 mm x 20 mm
Nunc Polyolefin Acrylate Sealing tape, Thermo Scientific	VWR	37000-548	To use with the 96-well  PCR plates for obtaining genomic DNA
Phire Animal tissue direct PCR Kit (without sampling tools)	Thermo Fisher	F140WH	For obtaining genomic DNA and performing PCR
Precision Melt Analysis Software	Bio Rad	1845025	Used for genotyping the mosquito DNA samples and analyzing the thermal denaturation properties of double-stranded DNA (see protocol step 3.3)
Single-channel pipettor	Gilson
Phire Animal tissue direct PCR Kit (without sampling tools)	Thermo Fisher	F140WH	For obtaining genomic DNA and performing PCR
SeqMan Pro	DNAstar Lasergene software		For multiple sequence alignment
Single-channel pipettor	Gilson