Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature
Published: May 31, 2023
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.
Divulgaciones
The authors have nothing to disclose.
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 |
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Citar este artículo
Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature. J. Vis. Exp. (Pending Publication), e21353, doi: (2023).