Gentotyping with Luna Probes and High Resolution Melting is a fast, cost effective and efficient alternative to other available genotyping methods such as TaqMan and direct sequencing.

Method Overview
This form of Genotyping uses asymmetric PCR and Luna (unlabelled) probes (20-40bp long that have been blocked at 3’ to prevent extension). With this method it possible to both genotype and scan for additional mutations in the larger amplicon.

Using asymmetric PCR, one strand of the DNA is produced in a greater quantity than the other. An unlabelled probe is designed to sit over the site of the mutation of interest and be complimentary to the over amplified strand. As the product is melted, the Luna Probe melts before the full product does. Generally the Luna Probe will melt in the temperature region of 50-65C. This is considerably cooler than the melt temperature of a product of over 200bp. Due to the small size of the Luna Probe, variations between samples give large variations in the melting profile. A typical variation is shown in Figure 1 below.

Using unlabelled probes one can both genotype the specific area under the probe and then scan the rest of the amplicon to look for other mutations. This way you can effectively get two tests from the single experiment.

See below for additional information in the form of papers and posters.

Figure 1: A typical set of data from a High Resolution Melting experiment using unlabelled Luna Probes. The blue shows the Homozygous mutation, the red shows the heterozygous mutation and the grey represents the wild type.


Additional Information:

1) Genotyping using unlabelled oligo probes - Application Note

2) Ultra Fast Mutation Discovery and Genotyping - Application Note

3) Genotyping Methods Compared - Poster