Figure 1.
Plus/plus genotyping assay design.
(A) Two-primer assays are suitable when common sequences flank a divergent sequence. The divergence may for example be due to (i) strain specific polymorphisms (such as microsatellite markers), or (ii) genomic incorporation of an orthologous DNA sequence and may generate products of differing size and/or sequence. (B) Three-primer assays are appropriate when the site of incorporation of exogenous DNA is known. The P1/P2 primers will specifically amplify a product from the modified locus by virtue of the exogenous DNA specific P2. The P1/P3 primers will only amplify a product from the wild type locus because PCR conditions are set to favour the short product and inhibit the amplification of the theoretically possible but much larger product from the altered locus. (C) Four-primer assays are useful when the location of the exogenous DNA is unknown and the exogenous sequence is unrelated to the mouse genome. The P1/P2 primers are unique to the exogenous DNA and P3/P4 can correspond to any unrelated region of the mouse genome. P: primer.
Figure 2.
Heterozygous animals have a melt profile distinct from that of either homozygote.
(A) Diagram representing the amplicons derived from genomic DNA that is homozygous for one of two differently sized alleles (Allele 1 or Allele 2) or heterozygous for the two alleles. The heterozygote PCR product will predominantly contain homoduplexes of either Allele 1 or Allele 2 because heteroduplex formation is less favourable due to length and/or sequence differences between the allele amplicons. (B–E) HRM data following a two primer PCR to amplify the SSLP marker microsatellite D8Mit155 from mice derived from crosses between the BALB/c and FVB inbred strains such that progeny may be homozygous for one of the parental alleles or heterozygous for the two alleles. D8Mit155 spans a TG dinucleotide repeat and produces an amplicon of 139 bp from the BALB/c genome and 151 bp from the FVB genome. The melting peak analysis readily distinguishes the heterozygote animals which exhibit two melting point peaks. Blue: PCR products derived from an animal homozygous for Allele 1, Grey: PCR products derived from an animal heterozygous for Allele 1 and 2, Red: PCR products derived from an animal homozygous for Allele 2.
Table 1.
Assay conversion.
Figure 3.
Small changes in fragment length or sequence are sensitively detected by HRMA.
(A) Agarose gel electrophoretic analysis of products derived from amplification of SSLP marker microsatellite D7Mit69 from 5 inbred strains of mice. The predicted allele sizes are: FVB; 230 bp, BL/6 and BALB/b; 236 bp, C3H and 129; 238 bp. (B) High Resolution Melt Analysis of the same products showing that the three allele sizes are readily discriminated using either Shifted melting curve or Difference curve analysis. Blue: FVB, Red: C3H and 129, Grey: BALB/b and BL/6. SM: 1 Kb+ DNA ladder, NTC: No template control.