Allele-Specific PCR (AS-PCR) - KASPâ„¢ (Kompetitive Allele Specific PCR) or PACEâ„¢ (PCR Allele Competitive Extension) or Allele-Specific Quantitative PCR (ASQ) based genotyping assay design for multiallelic discrimination of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci. The application is based on FastPCR software and provides professional facilities for genotyping assays design for SNP/InDel-specific KASP assay targeting primers (KASP Assay Mix) and the universal Master Mix.
Input Format: Sequence(s) can be pasted or uploaded as a file in FASTA format.
Primer design: | ||
Minimal length (nt): | ||
Maximal length (nt): | ||
Minimal Tm (°C): | ||
Maximal Tm (°C): | ||
Minimal Linguistic Complexity (%): | ||
SNP position on the 3'-terminus (1-12): |
Example 1. Formatting sequences for SNP:
>1
gctctctgtgtctgatccaagaggcgaggccagtttcatttgagcattaa [A/G] tgtcaagttctgcacgctatcatcatcaggggccgaggcttctctttgtt
>2
tcatattccagtttgggcgagttttaagataggtccgg [S] acagtctttgcggcgccaacgcgtctttctccagcagacagtccccggactgc
>3
tcatattccagtttgggcgagttttaagataggtccgg [C] acagtctttgcggcgccaacgcgtctttctccagcagacagtccccggactgc
Allele-specific PCR (AS-PCR) assays can be designed for discrimination of insertions/deletions (InDels) polymorphisms. The program imposes no size limit on length difference for InDels alleles, provided that all alleles can be aligned, and the alignment has a sufficient length of overlapping to target primers. Two, three or four allelic variants may be included for analysis to compute allele-specific primers (ASPs). Only differences between the variants must be shown within the brackets. One Allele-Specific Primer (ASP) will be computed for each of the input allelic variants. Also, one common primer (Universal Primer, UP) will be computed which targets a conserved region in all sequences. Input sequences should be in a FASTA format with differences between alleles placed within square brackets: [allele1/allele2/allele3/allele4].
Example 2. Formatting sequences for InDels:
>1
tcatattccagtttgggcgagttttaagataggtccgg [AG/] acagtctttgcggcgccaacgcgtctttctccagcagacagtccccggactgc
Example 3. Multi-nucleotide variants. Detection of multi-nucleotide variants (MNV) is possible using KASP. Sequence information for MNVs should be submitted using the [allele1/allele2] format:
>1
tgggcagcattagtagaagaaagtacaagaccgtgtgtagaggatactct [GATATACTTGAG/CAGTCC] agcagatagcgttggataggcgacaggattattggagcgccgtcgagaac
Example 4. Haplotypes. KASP assays can be designed to detect haplotypes of any size. Sequence information for haplotypes should be submitted using the [allele1/allele2] format. The [square brackets] should surround all SNPs that are part of the haplotype. Any nearby SNPs that are not considered to be part of the haplotype should be outside of the square brackets and should be identified using the appropriate IUPAC ambiguity code.
In this situation, the "SNP position on the 3'-terminus" value of 12 or greater can be increased.
>1
caaacaccaaactggtgagtcgtggtttacaacacgggagttcaaaactg
[TATCCGAATGACGAATGTTCACGTCCTTAAAC
/CATCCGAATCACGAATGTTCAGTTCCTTTAAG]
catcatgaaatgagtttagtttgggtggctcgtaagtagacataaggcac
Oligonucleotide specificity is one of the most critical factors for good PCR; optimal primers should hybridize only to the target sequence, especially when using complex genomic DNA as a template. Amplification problems can occur when primers anneal to repetitive sequences (retrotransposons, transposons or inverted tandem repeats). Alternative product amplification can also happen when primers are complementary to inverted repeats and produce multiple bands. However, the generation of inverted repeat sequences is exploited in two common generic DNA fingerprinting methods (RAPD).