PCR, Multiplex, and QF-PCR primer design & genotyping tool

This application offers advanced capabilities for designing primers across a wide range of PCR applications, including standard, inverse, multiplex, quantitative fluorescence (TaqMan and MGB-probe assay design), and bisulfite PCR. It also supports the development and validation of primer sets for genotyping single nucleotide polymorphisms (SNP) and insertions/deletions (InDel). Additionally, all individual tasks can be efficiently multiplexed for high-throughput analysis, such as fluorescence probe-based multiplex real-time qPCR assays.

Input format: Sequences can be pasted or uploaded as a file in FASTA format or retrieved sequence (NCBI’s accession, e.g. A02710) from NCBI’s "nuccore" nucleotide database or retrieved flanked sequence SNP sequences from the Ensembl database. One or multiple SNP/variant rsIDs (comma or space separated: rs1357617 rs2046361 rs717302 rs1029047 rs917118) were used to retrieve the surrounding sequence (±flank bases) and enter the species name (homo_sapiens, mus_musculus, rattus_norvegicus, bos_taurus, danio_rerio, arabidopsis_thaliana etc).

Size Limitations: the length of the query sequence and the size of the batch file are theoretically unlimited.

Primer design option		Multiplex PCR
Length range (nt):	-	Inverse PCR (circular sequence)
Tm range (°C):	-	TaqMan-probe design assay
Minimal linguistic complexity (%):		MGB-probe design assay
Variants of the 3'-end composition (5'-3'):		Overlaping primers
Range of PCR product size (bp):	-	C>>T bisulfite conversion
		Non-specific priming control
Forward primer tail (5'-3'):
Reverse primer tail (5'-3'):
Generate		Clear

To export the results: select all (Ctrl-A), copy (Ctrl-C) and paste (Ctrl-V) to Excel sheet.

Sequences are expected to be represented in the standard IUB/IUPAC nucleic acid codes are acceptable letters: B=(C,G,T), D=(A,G,T), H=(A,C,T), K=(G,T), M=(A,C), N=(A,C,G,T), R=(A,G), S=(G,C), V=(A,C,G), W=(A,T), Y=(C,T), U=Uracil, I=Inosine.

Variants of the 3'-end composition (5'-3'): the structure of the last nucleotides at the 3'-end of the primer, it can be specified by "N" for any pattern, or it can be encoded by one, two, three or more characters of standard or mixed letters. It is possible to specify one or more patterns (separated by spaces and of equal length): sws ssw sww wss www. For example, the pattern WSS corresponds to all variants of the 3'-end composition: acc acg agc agg tcc tcg tgc tgg.

Pre-designed list of primers/probes. Pre-designed primers/probes list is used for multiplexing with prior designed PCR primer/probe sets.

Linguistic sequence complexity (LC%) is a measure of the 'vocabulary richness' of a genetic text based on counting the number of possible combinations of nucleotides ("entropy" of the set of possibilities) to the theoretically possible one. This value for sequence is converted to percentages, 100% being the highest level.

The user can specify individually for each sequence location for both Forward and Reverse primers design using ‘[‘ and ‘]’ inside each sequence.

Optionally, use two ‘/.../’ signs for the start and end of the excluded region (this is possible multiple times).

All possible combinations of '[ Forward ]' with '[  Reverse ]' within the sequence(s):
1.  [SNP/InDel]
2.  [  ]  [  ]
3.  [  [  ]  ]

С >> T bisulfite conversion (bisulfite modified genome)

Sequence, design of specific PCR primers for in silico bisulphite conversion for both strands - only cytosines not followed by guanidine (CpG methylation) will be replaced by thymines:

5’aaCGaagtCCCCa-3'        5’aaCGaagtTTTTa-3'
  |||||||||||||     ->      ||||||:|::::| 
3’ttGCttCaggggt-5'        3’ttGCttTaggggt

Non-specific priming control

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).

Minor Groove Binders (MGBs) Hydrolysis Probes

Hydrolysis probes are typically labelled with a fluorophore reporter at the 5′end and a quencher [such as the Eclipse Dark Quencher (EDQ)] at the 3′end. During amplification, the DNA polymerase exonuclease activity cleaves off the reporter, allowing for signal unquenching and detection. Hydrolysis probes can be amended for increased binding stability or signal specificity. For example, MGBs selectively bind non-covalently to the minor groove, a shallow furrow in the DNA helix. Dual-labelled probes conjugated with MGB groups form extremely stable duplexes with single-stranded DNA targets, enabling shorter probe lengths and superior quenching.