Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

The overall goal of the following high resolution melt experiment is to increase the sensitivity of detection of mutant alleles present at low concentrations with an individual samples. This is accomplished by first preparing extracted DNA by diluting the template to the necessary volumes and concentrations. The second step is to prepare the assays with asymmetric proportions of the forward and reverse primer to increase the template probe product.

After preparing the reactions, the annealing temperature is set between the melting temperatures of the probe when bound to either the wild type or mutant template. The final step is to analyze the amplified products on the appropriate HRM platform. Ultimately mutant allele amplification bias and probe based asymmetric PCR allow more sensitive detection of challenging SNP mutation at low concentrations present in samples.

This increased sensitivity is useful for mutation surveillance in populations. The main advantage of this technique over existing methods like standard high resolution melting or tac man genotyping, is that it allows increased sensitivity for alleles present at low concentrations, and also allows genotyping of SNPs located proximal to each other in the genome. Though this method can provide insight into the spread of drug resistance in the malaria parasite.

It can also be applied to other systems such as surveillance of other infectious disease types such as HIV or detecting rare cancer variants in a population of cells. Demonstrating the procedure will be Caitlin Durphy. A technician from our laboratory.

Templates for high resolution melting or HRM are prepared from plasmodium falciparum samples obtained directly from the blood of patients participating in field sites studies. The samples can be stored on filter paper, on rapid detection tests or as pelleted red blood cells samples may also be culture adapted to grow in vitro. Some standard laboratory strains for analysis can be ordered from the malaria research and reference reagent resource center samples may be extracted from whole blood, red blood cells or filter paper, or used directly from pelleted red blood cells or culture for direct amplification from pelleted red blood cells.

First determine the para of the red blood cells using thin smear microscopy following the procedure from the Centers for Disease Control and Prevention. Red blood cells with paras above 0.5%are then diluted one to 400. In TE three microliters of diluted red blood cells will be used for each five to 10 microliter reaction due to between one variability positive and negative controls must always be included in HRM analyses.

Prepare 10 picograms to 10 nanograms per one microliter dilutions of plasmid or standards with sequence verified snip genotypes as positive trolls. Use PCR grade water as a no template negative control for the amplification reactions. Begin this procedure by preparing 10 x working stocks of the primers and probes.

The same protocol is applicable to assays using 96 well plates 384 well plates eight tube strips or glass capillaries. But for the purpose of this demonstration, only 96 well plates and glass capillaries will be used. This table shows recommended 10 x working stock concentrations as well as final concentrations for blocked probe based HRM genotyping.

Prepare reaction mixtures to each. Well add one microliter of each of the 10 x working stock forward and reverse primers and probes. Four to five microliters of the HRM master mix one microliter of template and PCR grade water for a total reaction volume of 10 microliters.

Do the same for each glass capillary. Cover the plates and glass capillaries. Use optical plate seals for plate based amplification and plastic caps for capillary based systems.

Make sure coverage is complete and plates and caps are completely sealed and secured to prevent evaporation. During amplification. Spin the samples to remove air bubbles.

Spin plates in a tabletop centrifuge for three minutes at 1, 800 times G.Spin glass capillaries in a pico fuge for 10 to 15 seconds. Place the reaction plate and glass capillaries in a thermal cycler run standard blocked probe or MAAB amplification protocols. After PCR amplification proceed to the melting analysis from the system software interface.

Melt the plate from 40 degrees Celsius to 80 degrees Celsius to produce both probe and amplicon melting peaks. The first step melting analysis on the light cycler four 80 is normalization from the negative derivative of normalized fluorescence with respect to temperature view. Move normalization bars to surround the probe melt region.

The probe melt region is the lower temperature of the two melting regions. The normalization bars should be at the base of the melt peaks. After normalization, select calculate groups to automatically assign samples to groups if necessary, manually reassign samples to specific groups.

Select samples that did not amplify or that have jagged melting peaks. Then go to new call and select negative. After selecting any remaining misclassified samples, go to new call, select the appropriate grouping and click apply change Assign genotypes for each group based on the standards.

After confirming that the computed groups are correct, select edit group names under the grouping tab. Enter the genotypes of the standards into the corresponding colored box. For example, if standard 3D seven has a known C genotype and is in group one, then all samples in group one have genotype C press results and genotypes are displayed beside samples.

Lastly, export the results as A TXT file for further sample population analysis. After the run finishes on the light cycler 2.0, record the sample names and positions under sample data Before starting the analysis label the negative controls and the genotypes of the melting standards. Begin the melt analysis by selecting analysis, choosing genotyping under melting curve analysis and pressing.Okay.

To increase the sensitivity of the auto grouping, select all of the samples so that the display in the melting curves plot. Slide the normalization bar to the upper boundary of the probe melting peaks. Confirm that the samples have been correctly grouped by individually selecting each group beneath group name.

Export the genotypes for each sample by selecting all of the samples, copying the data and pasting into a worksheet. The plot of the negative derivative of normalized fluorescence with respect to temperature is typically the most straightforward way to visualize melting peaks and determine genotypes. Setting the analysis window to just a probe region results in clear differentiation of peaks corresponding to specific SNPs.

Perfect matches result in higher melting peaks indicated in red. While SNP mismatches have lower melting temperatures indicated in gray when both alleles are present in a sample. Prob based HRM analysis represents both alleles as two peak curves shown in orange with peaks that match single allele samples shown in red and gray, progressively reducing the ealing temperature during amplification results in a bias towards the mutant allele represented by the left side peak in a poly genomic or poly allelic sample.

This mutant allele amplification bias results in HRM sensitivity to detect minor alleles present at less than 1%in poly genomic or poly allelic samples. While attempting this procedure, it's important to remember that sample quality is key. Minor differences in buffer concentrations can shift the HRM curves.

Using controls is a useful way to standardize results. After watching this video, you should have a good understanding of how to use prob based asymmetric PCR and mutant allele amplification bias to accurately and sensitively genotype malaria SNPs from a variety of sample types.