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Prim-SNPing: A primer designer for cost-effective SNP genotyping

Article in BioTechniques · June 2009

DOI: 10.2144/000113092 · Source: PubMed

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 Short Technical Reports

Short Technical Reports SNP genotyping with fluorescence polar-

ization detection (2), the Invader assay (3),
DNA microarray (4,5), and pyrosequencing
(6). Currently, most of these methods are
prohibitively expensive.
Prim-SNPing: a primer designer for Among these methods, PCR-RFLP has
become one of the more commonly used
cost-effective SNP genotyping methods for genotyping for genetic associ-
ation studies in laboratories. PCR-RFLP
requires PCR amplification and restriction
Hsueh-Wei Chang1,2,3, Li-Yeh Chuang4, Yu-Huei Cheng5, Yu-Chen enzyme digestion before electrophoresis (7).
Hung1, Cheng-Hao Wen1, De-Leung Gu1, and Cheng-Hong Yang5 However, DNA digestion requires 3–24 h,
1Department of Biomedical Science and Environmental Biology, Kaohsiung
depending on the applied restriction enzyme.
Recently, a novel method, PCR with
Medical University, Taiwan, 2Graduate Institute of Natural Products, confronting two-pair primers (PCR-CTPP),
College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, has been developed to perform SNP
3Center of Excellence for Environmental Medicine, Kaohsiung Medical genotyping without DNA product digestion
(8–11). If the digestion step can be skipped,
University, Kaohsiung, Taiwan, 4Department of Chemical Engineering, the SNP genotyping process is greatly accel-
I-Shou University, Taiwan, and 5Department of Electronic Engineering, erated. However, it has been suggested that
National Kaohsiung University of Applied Sciences, Taiwan the melting temperature (Tm) range for the
primer design in PCR-CTPP is limited (9).
BioTechniques 46:421-431 (May 2009) doi 10.2144/000113092 This limitation reduces the practical use of
Keywords: primer design; SNP genotyping; RFLP; mutagenic primer; PCR-CTPP PCR-CTPP without computational help.
Supplementary material for this article is available at www.BioTechniques.com/article/113092. Although the PCR-RFLP and PCR-
CTPP methods are cost-effective, the devel-
opment of computational algorithms for their
Many kinds of primer design (PD) software tools have been developed, but primer design is necessary for researchers
most of them lack a single nucleotide polymorphism (SNP) genotyping service. without a background in SNP genotyping.
Here, we introduce the web-based freeware “Prim-SNPing,” which, in addition Fortunately, many inherent primer design
problems have been solved, including the
to general PD, provides three kinds of primer design functions for cost-effective mutagenic primer problem (12,13), primer-
SNP genotyping: natural PD, mutagenic PD, and confronting two-pair prim- dimer and hairpin structures by AutoDimer
(14), and secondary structure and optimized
ers (CTPP) PD. The natural PD and mutagenic PD provide primers and re- downstream genotyping applications by
striction enzyme mining for polymerase chain reaction–restriction fragment DFold (15). SNP Cutter (16) provides both
of length polymorphism (PCR-RFLP), while CTPP PD provides primers for natural PD and mismatch (mutagenic) PD
for PCR-RFLP genotyping with online
restriction enzyme–free SNP genotyping. The PCR specificity and efficiency of input but email output for the result. Conse-
the designed primers are improved by BLAST searching and evaluating second- quently, we have enough information to
develop a well-rounded primer design tool,
ary structure (such as GC clamps, dimers, and hairpins), respectively. The length which can be coupled with previously intro-
pattern of PCR-RFLP using natural PD is user-adjustable, and the restriction duced software, SNP-RFLPing (17) for SNP
sites of the RFLP enzymes provided by Prim-SNPing are confirmed to be absent genotyping.
In this study, we introduce Prim-SNPing,
within the generated PCR product. In CTPP PD, the need for a separate diges- an improved software tool for primer
tion step in RFLP is eliminated, thus making it faster and cheaper. The output of design and RFLP enzyme mining for cost-
Prim-SNPing includes the primer list, melting temperature (Tm) value, GC per- effective SNP genotyping, which supports
both online input and output. Free format
centage, and amplicon size with enzyme digestion information. The reference sequence inputs and reference SNP cluster ID
SNP (refSNP, or rs) clusters from the Single Nucleotide Polymorphism data- number (SNP ID rs#) inputs are acceptable.
We also developed a novel primer design
base (dbSNP) at the National Center for Biotechnology Information (NCBI), function “CTPP PD” for SNP genotyping,
and multiple other formats of human, mouse, and rat SNP sequences are accept- which omits the digestion step for restriction
able input. In summary, Prim-SNPing provides interactive, user-friendly and enzymes. Therefore, Prim-SNPing provides
user-friendly and cost-effective primer
cost-effective primer design for SNP genotyping. It is freely available at http:// design for SNP genotyping. The software is
bio.kuas.edu.tw/prim-snping. available for free at http://bio.kuas.edu.tw/
prim-snping.

chain reaction–restriction fragment

Introduction of length polymorphism (PCR-RFLP)
Materials and methods
To date, several single nucleotide polymor- analysis, DNA sequencing, Taqman Implementation
phism (SNP) genotyping approaches have probes, and kinetic PCR. Other, more Prim-SNPing, a web-based interface, was
been reviewed (1), including polymerase recently developed approaches include designed and implemented under the
Vol. 46 | No. 6 | 2009 421 www.BioTechniques.com
Short Technical Reports

SQL server database system. Java server the MySQL format and a local database Design, (4) Annealing Check, (5) RFLP
pages and Java applets are used to input copy, respectively. We plan to update Analysis, and (6) Output.
data and process files between the user these databases annually.
and the application, and to parse the data, Input module
respectively. The database structure is Program workflow Four kinds of primer design types (i.e.,
mainly set up by REBASE v. 607 (http:// The schematic program workflow of general PD, natural PD, mutagenic PD
rebase.neb.com) (18) and NCBI dbSNP Prim-SNPing (Figure 1) consists of six and CTPP PD) are provided. Except for
Build 125 (19), and is transformed into modules: (1) Input, (2) Query, (3) Primer the general PD, all other forms of PD in
Prim-SNPing provide the primers for SNP
genotyping in a cost-effective manner.
Sequence input types include SNP ID rs#
feed-in, sequence copy-and-paste, and text
file upload. Primer design constraints are
shown in detail in Figure 1.

Query module
When a user inputs the NCBI SNP ID rs#,
the sequence information for the targeted
SNP is retrieved from the local NCBI
dbSNP database. The contig position
is retrieved from NCBI’s Reference
Sequence (RefSeq) (20) to increase the
length of short flanking sequences, which
are usually provided in NCBI dbSNP.
The Prim-SNPing system can provide a
maximum flanking sequence length of
1000 bp for each SNP. This service for
retrieval of flanking sequence improves
the system’s flexibility for primer design.
Figure 1. Flowchart of web-based Prim-SNPing. Three kinds of functions are incorporated into the
input module of the Prim-SNPing system: the primer design (PD) type (general PD, natural PD, Primer design module
mutagenic PD and CTPP PD), the sequence input type (rs#, sequence and file input), and the The primer design module provides
primer design constraints, as indicated. The query module provides the SNP FASTA sequence and primers based on the settings of the input
flanking sequence nearby the SNP using rs# search via NCBI dbSNP (19). In a next step, the flank-
ing sequence of the SNP is fed into the primer design module for usage with the four PDs. Subse-
module (Figure 1). Default parameters
quently, the annealing check and RFLP analysis modules provide further analysis if selected. In the such as primer length (∼18–26 bp), primer
RFLP module, the SNP-containing sequences are transferred to a local database downloaded from length difference (5 bp), GC proportion
REBASE (18), and then the RFLP availability for the sense and antisense sequences is determined. (∼40–60%), Tm calculation, hairpin,
Finally, the output module displays the RFLP enzyme and primer information as indicated. Tm, melt- and GC clamp of primers are similar to
ing temperature; Tm-diff, Tm difference.
the ones used in the literature (21–25).
In general PD, primers are designed only
Table 1. Comparison of the Features of Primer Software Tools
for regular PCR without SNP genotyping
consideration. For natural PD, mutagenic
Primer3-

Primer Z

SNPbox

SNPing
WASP

Prim-

PD, and CTPP PD, the primer infor-

Plus

PDA

mation for SNP genotyping is provided.

Natural primers are designed for targeted
SDP rs#ID • • •
SNPs with available RFLP enzymes. In
Sequence- text • • • •
Input

the case of a SNP without RFLP enzyme,

Sequence-FASTA • • • •
the mutagenic primers are designed by
File upload • • • •
changing the nucleotide beside the SNP
in order to determine its RFLP avail-
Secondary
structure

Hairpin, Dimer, GC clamp • • • • • • ability. Once the availability is confirmed,

the opposite primer is designed according
to a user-defined length range. In CTPP
%CG, Tm • • • • • •
PD, the confronting two-pair primers are
Na, Mg & Salt Con. • • • •
designed to create different lengths of the
Primer length • • • • • •
Parameter

PCR product corresponding to its SNP

Setting

PCR product size • • • • • •

Extension of SNP flanking sequences • • • •
genotype. The principle of PCR-CTPP (9)
BLAST for primers • •
and the locations of four essential primers
Ratio for RE-digested RFLP length •
are illustrated in Figure 2.
Regular primers • • • • • •
RFLP-natural primers •
Annealing check module
Output

The quality of the primer set is determined

typing
geno-
SNP

RFLP-mutagenic primers •
by (i) GC clamp, (ii) dimer check, (iii)
CTPP primers •
hairpin check, and (iv) BLAST specificity

Vol. 46 | No. 6 | 2009 422 www.BioTechniques.com

Short Technical Reports

adjusted. In Figure 3G, the range for the

mutagenic base of the SNP is set to ±5 bp
for mutagenic PD. The annealing check
module shown in Figure 3H provides
highly specific and optimal primer criteria
for improving the quality of the designed
primers.

Output data
By clicking the corresponding box in
Figure 4A, the designed primers can be
sorted by Tm difference and processed by
the BLAST function. The user-defined
primer conditions are shown in Figure
4B. Ten primers are provided for each
primer design (PD) in Prim-SNPing
(only one or two representative primers
are shown in Figure 4). In Figures 4C
and 4D, both the commercial and
Figure 2. Introduction of the PCR-CTPP method (modified from References 9 and 10). In the example, non-commercial restriction enzymes
the PCR-CTPP is designed to confront two-pair primers like F1, F2, R1, and R2, where F2 and R1 for SNPs in the sense (input) strand are
are opposite to the alternative nucleotide of the same SNP in both sense and antisense strands, mined from the local REBASE database
respectively. If the SNP is A/G in the sense strand and the primer F2 only covers SNP-A (sense
strand), the nucleotide in primer R1 is SNP-C (antisense strand) for its corresponding SNP-G in the (18) incorporated in Prim-SNPing. The
sense strand. (B) Primers F1/R2 are used for PCR as a positive control designed for both alternative BLAST function of a primer is acces-
nucleotides of the SNP (i.e., SNP-A and SNP-G). Primers F1/R1 and F2/R2 are preferentially used sible by clicking on it individually. The
for SNP-A and SNP-G, respectively. When electrophoresis is performed after the PCR, the AA type BLAST time for a primer is rather long
should be positive for PCR using F1/R1 and F2/R1 primers and the GG type should be positive for due to the short primer length. In Figures
PCR using F2/R2 and F2/R1 primers. For heterozygous type AG, PCR using F1/R1, F2/R2, and F2/
R1 primers should all be positive. Prim-SNPing provides primers to create different lengths for these
4C, 4D, and 4E, the Tm difference for the
three PCR reactions. Accordingly, the SNP genotype data obtained by PCR-CTPP primers contains primer designed in Prim-SNPing is very
additional information. small, ensuring a highly effective PCR
reaction. The primer sequence, product
for the secondary structure and specificity flanking sequence is adjustable in a range size, GC number, GC percentage, Tm,
of primers (Figure 1). These tests ensure of up to 1000 bp. The SNP flanking Tm difference, and primer length are
quality control of the designed primers. sequences provided by Prim-SNPing provided. In Figure 4D (mutagenic PD),
are pre-computed; they are longer than the position of the changed nucleotide
RFLP analysis module the SNP FASTA sequences provided (blue color) is adjustable. Figure 2 serves
The RFLP analysis module is constructed by NCBI dbSNP. This increased length as a reference to determine the relative
by REBASE as described (17). Natural is beneficial for the primer design. location of the primers designed in CTPP
and mutagenic primers are processed, For example, the length of SNP ID PD (Figure 4E). In brief, the primers F2
and the RFLP analysis module is used rs4930098 (http://www.ncbi.nlm.nih. and R1 as described (9) represent the
to verify the availability of restriction gov/SNP/snp_ref.cgi?rs=4930098) is default SNP-containing primers that
enzymes in the SNP sequence. 601 bp. When updating this SNP ID, confront each other for the alternative
Prim-SNPing provides a maximum of nucleotide of the SNP. The Prim-SNPing
Output module 1000 bp. The output results are reported system narrows the Tm difference down
Finally, information from the primer online and/or via email (Figure 3D). to within 1°C via computation, a value in
design modules, the RFLP analysis Before the primer design output, accordance with requirements listed in
module, and the annealing check module Prim-SNPing provides further analysis the PCR-CTPP literature (9). Finally, the
is integrated in the output module and using the advanced options listed in system provides the BLAST check for the
made available online and via email. Figure 3, E–G and an annealing check sequences of primers rather than BLAST
as shown in Figure 3H. Figure 3E shows check for the sequence of PCR amplicon
the common part for all primer designs (Figure 4F).
Results and discussion provided by Prim-SNPing. In principle,
Input data PCR-RFLP for SNP genotyping depends comparison of some primer design
Input data for Prim-SNPing is line-fed on the restriction enzyme availability software tools with the Prim-SNPing
through the web interface for the human, occurring in only one of the alternative Many primer design tools have been
rat or mouse SNP genotyping assay. The nucleotides of the targeted SNP (17). developed. Primer 3 (25) is one of the
input mode contains sequence and file Therefore, the lengths of the undigested earliest and most commonly used primer
inputs like the ones shown in Figure 3A. and digested PCR products vary design software tools. Primer Design
When clicking on the input button, the depending on the location of the primers. Assistant (PDA) (26) is a web interface
sequence data with the SNP in [dNTP1/ Prim-SNPing provides a novel function primer design service, which incorporates
dNTP2] or IUPAC format is fed into for the user-defined RFLP pattern for thermodynamic theory to evaluate the
the sequence window for primer design natural PD (Figure 3F). The “Ratio for fitness of primers. Recently, Primer3Plus
(Figure 3B). NCBI SNP ID rs# input RE (restriction enzyme)–digested RFLP (27), an enhanced web interface of
is also acceptable (Figure 3C) and the length” is set to 3:1 by default but can be Primer 3, was introduced. WASP (28)

Vol. 46 | No. 6 | 2009 424 www.BioTechniques.com

Short Technical Reports

of Primer Z, none of these tools provides

a BLAST function. Therefore, these
tools cannot check for possible multiple
annealing in the genome to prevent
primers from nonspecific binding to
genomic DNA. The features of the primer
design software tools are compared, as
shown in Table 1.
SNPicker (31) is another primer design
tool for SNP genotyping, which uses
REBASE (18) to automatically scan for
all possible designs of mutagenic primers.
Unfortunately, it is not web-based. FESD
(32), on the other hand, identifies flanking
sequences for SNP IDs, but a primer
design function is not included. Given
the lack of functions in these individual
software applications, it is evident that
there is a need for the development of
an entirely online-based and integrated
software package for SNP genotyping
with primer design functions.
In this study, we focus on the
software development of cost-effective
SNP genotyping, for which input
and output are entirely online-based.
The advanced options (Figure 3E)
and annealing check function (Figure
3H) vastly enhance the quality of the
designed primers. Acceptable sequence
formats for Prim-SNPing are copy-and-
paste, file upload in a freely selectable
Figure 3. Input data for Prim-SNPing. (A) Input mode. Sequence and file input are acceptable for format, and SNP input as [dNTP1/
all primer tools in Prim-SNPing. (B) IUPAC format or [dNTP1/dNTP2] format (e.g., [A/C] or M) for dNTP2] or IUPAC code. Prim-SNPing
SNPs within the input sequence are acceptable. When inputting data, typing empty space does not also provides SNP ID rs# input in NCBI
interfere with the design. SNPs for human, mouse and rat genomes are included. (C) SNP ID input dbSNP (19) for natural PD, mutagenic
for primers used in SNP genotyping. The SNP FASTA sequence for the SNP flanking region is eas- PD, and CTPP PD. In SNP Cutter (16),
ily uploaded by clicking the “enter” box after keying in the SNP rs# ID of NCBI dbSNP. The SNP
flanking length is adjustable in the range of 500–1000 bp. (D) A report of the results is provided
a special format is required for the SNP
online and by email. (E) Common constraints for primer design. Several common criteria for primer input sequence. SNP Cutter provides
design in general PD, natural PD, mutagenic PD and CTPP PD are included. The system uses default online input, but results output is only
and user-defined settings or mixed selections of both. (F) Special input constraint of natural PD. available via email. The performance
The length of RFLP fragments is user-adjustable. Information similar to gel analysis is provided. (G) of Prim-SNPing–designed primers for
Special input constraint of mutagenic PD. The position of the artificially changed nucleotide in the the general PD, natural PD, mutagenic
mutagenic primer is selectable. (H) Annealing check for all PD tools in Prim-SNPing.
PD, and CTPP PD were all successfully
tested for at least two different PCR
is a web-based allele-specific PCR assay primer design tool for promoters, exons, amplicons from different genes for each
designing tool for detecting SNPs and and human SNPs. However, none of these PD (data not shown).
mutations. SNPbox (29) is a modular tools contains a SNP genotyping service, As an innovation, the length
software package for large-scale primer such as restriction enzyme mining for pattern of the RFLP using natural PD
design. Primer Z (30) is a streamlined RFLP genotyping. With the exception is user-adjustable (Figure 3F), and the
provided RFLP enzymes for a target
SNP are confirmed to be absent within
the generated PCR product. These
BIOMEDICAL Optics. features play an important role in SNP
genotyping. If the system-provided RFLP
t UV Lenses – Wide Selection of Coatings enzymes occur within the PCR product
using designed primers, then the digested
t UV Filters – High Transmission OD 6 Rejection patterns are changed. In other words,
the PCR products are cut at more than
t Request your FREE catalog! only one site by the restriction enzyme,
resulting in an undesirable complexity of
the RFLP genotyping process.
800.363.1992 | www.edmundoptics.com Some characteristics of the
PCR-CTPP method have been reported

Vol. 46 | No. 6 | 2009 426 www.BioTechniques.com

Short Technical Reports

(grant nos. NSC97-2311-B-037-003-

MY3, 97-2622-E-151-008-CC2, NSC-
96-2221-E-214-050-MY3, NSC96-
2311-B037-002, NSC96-2622-E214-
004-CC3, 96-2622-E-151-019-CC3, and
KMU-EM-97-2.1a).
The authors declare no competing
interests.

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FESD: a Functional Element SNPs Database in human. Nucleic Acids
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employment opportunities, and events.

Received 3 May 2008; accepted 7 January 2009. Advance Online Publication (AOP)
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Vol. 46 | No. 6 | 2009

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