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Theoretical and Applied Genetics

, Volume 130, Issue 3, pp 597–607 | Cite as

An innovative SNP genotyping method adapting to multiple platforms and throughputs

  • Y. M. Long
  • W. S. Chao
  • G. J. Ma
  • S. S. Xu
  • L. L. QiEmail author
Original Article

Abstract

Key message

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility.

Abstract

Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3′ regions to significantly increase the amplification specificity of the two alleles and tailed at 5′ ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion–deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Keywords

Priming Element Individual SNPs Size Separation Common Reverse Primer Amplification Yield 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Introduction

Many of the molecular markers widely used today in agriculture, medicine, and forensics are based on the single nucleotide polymorphisms (SNPs) that are highly distributed in the genome of every organism. Recent advances in sequencing technology have accelerated the discovery of vast variations in DNA sequences by many genome sequencing projects and association mapping by genotyping by sequencing (GBS). Currently there are two types of important technologies, microarray-based and PCR-based, employed in genotyping of SNPs. The multiplex chip-based technology for genome-scale genotyping of SNPs has made great progress in the past two decades and has been exploited and applied to genetic studies and/or medicine applications across a large number of species including human, animals, plants, and microbes.

On the contrary to the well-established platforms for genome-scale genotyping of SNPs, PCR-based technology for genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs. Currently there are four major PCR-based approaches for genotyping of individual SNPs (Syvänen 2001): T m/T a (melting temperature/annealing temperature)-based genotyping approaches including allele-specific probe (AS-probe) hybridization (Livak et al. 1995; Tyagi and Kramer 1996; Tyagi et al. 1998; Livak 1999; Whitcombe et al. 1999) and high-resolution melt (HRM, Taylor et al. 2010), allele-specific PCR (Newton et al. 1989; Myakishev et al. 2001; Semagn et al. 2014), single nucleotide sequencing (mini-sequencing, Syvänen et al. 1990; Pastinen et al. 1997), and restriction site cleavage (CAPS, Konieczny and Ausubel 1993; Neff et al. 1998). Each of these approaches has its own advantages but each also has at least one major disadvantage (Table S1). For example, mini-sequencing provides high accuracy and detection rate of SNPs, but is slow as well as being expensive because of the cost of the instrument, reagents, and labor. The disadvantages in each of these approaches hamper their widespread use. Another problem is that the different approaches require different genotyping platforms. The researchers who employ different approaches cannot share SNP genotyping conditions, and genotyping platform replacement requires primer redesign that is costly to laboratories. In traditional genotyping of indels (insertion–deletion polymorphisms), allele calling by size separation is time-consuming and labor-intensive.

To overcome the limitations of current technologies for genotyping of individual SNPs, this study aimed to develop a new genotyping method which combines all the major advantages (i.e., high accuracy, flexible throughputs, simple assay design, and low operational costs) of a number of currently used SNP genotyping approaches and is compatible with a variety of platforms.

Materials and methods

Plant materials

Two rice (Oryza sativa L.) inbred lines (93-11 and Nipponbare), six sunflower (Helianthus annuus L.) inbred lines (HA 89, HA 412-HO, RHA 415, RHA 373, RHA 280, and RHA 801), and the 96 F2 plants from the cross between Aegilops tauschii Cosson accessions AL 8/78 and CIae 25 were used in this study. The DNAs from rice, Ae. tauschii, and sunflower represent DNA sources from small genome, complex genome, and complex genome rich in polysaccharides and polyphenols, respectively. The genomic sequences of rice inbred lines 93-11 and Nipponbare are available in NCBI (International Rice Genome Sequencing Project 2005). The DNA sequence variations between Ae. tauschii accessions AL 8/78 with CIae 25 were identified by Sanger sequencing of PCR fragments. The six sunflower inbred lines were maintained in USDA-ARS, Fargo, ND. The SNP sequences of sunflower were downloaded from Bachlava et al. (2012).

STARP (semi-thermal asymmetric reverse PCR) conditions

STARP was carried out in a total volume of 10 µl mixture containing 1 × NH4 + buffer (16 mM (NH4)2SO4 and 67 mM Tris–HCl, pH 8.3 at 25 °C), 0.8 M betaine, 0.04% (W/V) bovine serum albumin (BSA), 1.5 mM MgCl2, 50 µM of each dNTP, 200 nM common reverse primer (see primer design in results), 200 nM of each of priming element-adjustable primers (PEA-primer 1 and PEA-primer 2), and 40 nM of each of asymmetrically modified allele-specific primers (AMAS-primer 1 and AMAS-primer 2), 1 U of Taq DNA polymerase (without 3′ → 5′ exonuclease activity), and genomic DNA in the range from 10 to 100 ng. All the PCR mixtures were set on ice, mixed well, and spun down. PCR was performed with initial denaturation at 94 °C for 3 min, followed by 6 cycles of 2-step touchdown PCR program starting at 94 °C for 20 s and then 56 °C for 2 min, with the annealing/extension temperature (T a/e) being decreased by 1 °C per cycle. This touchdown PCR program was immediately followed by another 2-step PCR program of 94 °C for 20 s and then at 62 °C for 2 min with 32–36 cycles for gel-based size separation or 42–46 cycles for gel-free fluorescence signals. The amplification was completed with a 2-min extension at 62 °C.

Results

Primer design

To detect the two alleles (allele 1 and allele 2) of a SNP, each PCR in the STARP assay requires two universal priming element-adjustable primers (PEA-primer 1 and PEA-primer 2) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primer 1 and AMAS-primer 2) and their common reverse primer. STARP behaves as 2-plex PCR. Allele 1 is specifically amplified using PEA-primer 1 coupling with AMAS-primer 1 in combination with the reverse primer, and Allele 2 is specifically amplified using PEA-primer 2 coupling with AMAS-primer 2 in combination with the common reverse primer. The two AMAS-primers are designed to specifically amplify their target alleles and provide priming sites for PEA-primers. The two PEA-primers are used as universal primers in all genotyping assays for stringent targeting of the PCR fragments generated by the AMAS-primers with similar PCR efficiencies and for flexible SNP detection using either gel-based size separation or gel-free fluorescence signals. The common reverse primer is used to synthesize complements of both alleles.

Each of the PEA-primers consists of a 5′-stem element, a PCR blocker (Spacer 9 or Sp 9, triethylene glycol), and a distinct priming element harboring a 3′-stem element. In addition to these basic elements, an insertion (AGAG-3′) is introduced between the 5′-stem element and the PCR blocker of PEA-primer 2 (Fig. 1a; Table 1). This additional insertion generates the difference in length between the ultimate PCR fragments of the two alleles and thus supports bi-allelic discrimination by size separation. During the PCR, each PEA-primer forms a hairpin structure at low temperature to block its priming element from spurious hybridization and dimer formation (Fig. 1b). The hairpin structure is linearized at higher temperature to expose the priming element for stringent amplification (Fig. 1a, c; Table 2). This versatile structure also supports bi-allelic discrimination by fluorescence signals. The PCR blocker prevents DNA polymerase from reading through the hairpin structure. This enhances PCR efficiency and specificity. The two priming elements in the PEA-primers contain an identical six-base oligonucleotide (TATGAC-3′) at their 3′ regions, ensuring that PEA-primer 1 and PEA-primer 2 are always extended with similar efficiencies in genotyping of different SNP loci. Under standard PCR conditions, the amplification efficiency of PEA-primer 1 is 80–90% of that of PEA-primer 2 (Fig. 1d). This minor difference can be used to rectify the difference in amplification efficiencies between the two AMAS-primers, as described in the following.
Fig. 1

Structure and characterization of PEA-primers. a Linear structure of the two dual-labeled PEA-primers. PEA1-QFAM indicates Dabsyl (Quencher) and FAM (fluorophore)-labeled PEA-primer 1, and PEA2-QHEX indicates Dabsyl and HEX (fluorophore)-labeled PEA-primer 2. b Hairpin structure of the two dual-labeled PEA-primers. The hairpin structure blocks the priming element and brings the fluorophore and the quencher into close proximity, and fluorescence is quenched. c Melting peak of the two dual-labeled PEA-primers. The melting peaks of PEA1-QFAM and PEA2-QHEX were obtained using the standard PCR mix with the exception of Taq. Their T m values are 59 and 64 °C, respectively. Both PEA-primers form the most stable hairpin structures at 33 °C. d Comparison of the amplification efficiencies between the two PEA-primers. Nine genome-derived sequences each were attached with tail 1 and tail 2 at its 5′ end, respectively, to generate nine pairs of forward primers. Each pair of these forward primers, two IRDye® 700-labeled PEA-primers (PEA1-700 and PEA2-700), and the reverse primer were used to perform PCR reactions. Each gel shows PCR fragments generated in the same genomic DNA with three replicas under the standard PCR conditions. The small (low) and big (upper) bands represent PCR fragments generated by PEA1-700 and PEA2-700, respectively. These nine gels show the similar trends in PCR efficiencies between the two PEA-primers

Table 1

Detailed sequence information of PEA-primers

Unit name

Sequence

PEA-primer 1

AGCTGGTT-Sp9-GCAACAGGAACCAGCTATGAC-3′

5′-Stem element in PEA-primer 1

AGCTGGTT-3′

Priming element 1

GCAACAGGAACCAGCTATGAC-3′

3′-Stem element in PEA-primer 1

AACCAGCT-3′

PEA-primer 2

ACTGCTCAAGAG-Sp9-GACGCAAGTGAGCAGTATGAC-3′

5′-Stem element in PEA-primer 2

ACTGCTCA-3′

Priming element 2

GACGCAAGTGAGCAGTATGAC-3′

3′-Stem element in PEA-primer 2

TGAGCAGT-3′

Insertion in PEA-primer 2*

AGAG-3′

PCR blocker (spacer 9 or Sp 9)

Triethylene glycol

PEA1-QFAM

5′d FAM-AGCTGGTT-Sp9-GCAACAGGAACCAGC-T(Dabsyl)-ATGAC-3′

PEA2-QHEX**

5′d HEX-ACTGCTCA-Sp9-GACGCAAGTGAGCAG-T(Dabsyl)-ATGAC-3′

PEA1-700

5′d IRDye® 700-AGCTGGTT-Sp9-GCAACAGGAACCAGCTATGAC-3′

PEA2-700

5′d IRDye® 700-ACTGCTCAAGAG-Sp9-GACGCAAGTGAGCAGTATGAC-3′

PEA1-800

5′d IRDye® 800-AGCTGGTT-Sp9-GCAACAGGAACCAGCTATGAC-3′

PEA2-800

5′d IRDye® 800-ACTGCTCAAGAG-Sp9-GACGCAAGTGAGCAGTATGAC-3′

* The AG motif numbers can be changed to adjust the difference in length between the ultimate PCR fragments of the two alleles

** The insertion in PEA2-QHEX was removed in the present study

Table 2

Characterization of PEA-primers

TPrimer name

Priming element

Hairpin structure

Fluorescence intensity (RFU)c

Length (base)

GC (%)

T m value (°C)a

ΔG (kcal mol−1)b

Predicted T m (°C)b

Measured T m (°C)c

PEA1-QFAM

21

52.4

59

−6.58

63

59

3055

PEA2-QHEX

21

52.4

59

−6.51

62

64

2769

aThe T m values of the priming elements were calculated with the primer-BLAST suite (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)

bThe parameters of the hairpin structures were calculated with OligoAnalyzer 3.1 (https://www.idtdna.com/calc/analyzer)

cThe fluorescence signals (RFU) were measured in the standard PCR mix with the exception of Taq, and the minimum RFU was recorded at approximately 33 °C

The pair of AMAS-primers is designed to specifically amplify their target alleles with similar efficiencies. Each AMAS-primer consists of a 5′ tail and a 3′ AMAS-sequence (Fig. 2a). Tail 1 and tail 2 in the AMAS-primers have the same sequences as priming element 1 and priming element 2 in the PEA-primers (Fig. 2b), respectively. The 3′ AMAS-sequences originate from the known allele-specific (AS-) sequences with the AS-nucleotide (SNP) at its 3′ terminus (Fig. 2c). To employ standard PCR conditions for all genotyping assays, the length of the two AS-sequences must be the same, and their melting temperatures (T m) should range from 54 to 58 °C as calculated with the Primer-BLAST suite. (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). This T m range is applied because of the correlation between the predicted T m value of the AS-sequence and the optimal annealing/extension temperature (T a/e) range of its corresponding AMAS-primer (Table S2, see discussion). To achieve high specificity of the initial amplification of both alleles, the 3rd nucleotide in one AS-sequence and the 4th nucleotide in the other AS-sequence from the 3′ terminus are substituted based on the following principle: A is changed to (→) C, T → C, G → A, and C → T. Thus, the two asymmetrically modified AS-sequences (AMAS-sequences) contain three different nucleotides at the 1st, 3rd, and 4th positions from the 3′ terminus (Fig. 2a). The difference in extension efficiencies between these two AMAS-sequences can be rectified by assigning a tail according to the SNP types shown in Table 3.
Fig. 2

AMAS-primer design using [C/G] SNP as an example. a AMAS-primer structure. The red hexagons and green solid circles indicate AS-nucleotides (SNP) and substituted nucleotides, respectively. b Graphic illustration of tails. c AMAS-primer design procedure. Based on the SNP type presented in Table 1 in text, allele C is designated as allele 1 (left graph, Step 1) and G as allele 2 (right graph, Step 1). The allele-specific nucleotides C and G preceded by its upstream flanking sequence are selected as AS-sequence 1 and AS-sequence 2 (Step 2), respectively. The length of the two AS-sequences is the same, and their T m values range from 54 to 58 °C. The 4th nucleotide in AS-sequence 1 and the 3rd nucleotide in AS-sequence 2 from 3′ terminus are substituted based on the principle mentioned in text (Step 3, green solid circle). And then, tail 1 and tail 2 are attached on the AS-sequence 1 and AS-sequence 2 at 5′ end, respectively, to complete design of the pair of AMAS-primers (Step 4). If the downstream flanking sequence of SNP site is chosen to design AMAS-primer, both allelic sequences are converted into their reverse-complement counterparts to design AMAS-primers

Table 3

AMAS-primer design for all six types of SNPs

SNP type

AS-nucleotide

Designated allele name

Assigned tail*

Designated AMAS-primer name

Nucleotide position from the 3′ end for substitution

[C/G]

C

Allele 1

[Tail 1]

AMAS-primer 1

4th

G

Allele 2

[Tail 2]

AMAS-primer 2

3rd

[C/T]

C

Allele 1

[Tail 1]

AMAS-primer 1

4th

T

Allele 2

[Tail 2]

AMAS-primer 2

3rd

[C/A]

C

Allele 1

[Tail 1]

AMAS-primer 1

3rd

A

Allele 2

[Tail 2]

AMAS-primer 2

4th

[G/T]

G

Allele 1

[Tail 1]

AMAS-primer 1

4th

T

Allele 2

[Tail 2]

AMAS-primer 2

3rd

[G/A]

G

Allele 1

[Tail 1]

AMAS-primer 1

3rd

A

Allele 2

[Tail 2]

AMAS-primer 2

4th

[T/A]

T

Allele 1

[Tail 1]

AMAS-primer 1

3rd

A

Allele 2

[Tail 2]

AMAS-primer 2

4th

[Tail 2] = GACGCAAGTGAGCAGTATGAC-3′

* [Tail 1] = GCAACAGGAACCAGCTATGAC-3′

The complements of both alleles are synthesized using a common reverse primer that is also designed with the Primer-BLAST suite. To prevent spurious amplifications, the reverse primer should locate in the single-copy region in genome or have more than two bases at 3′ end which are different from non-target sequences, The T m value of reverse primer should range from 58 to 62 °C to match that of the priming elements in the PEA-primers (Table 2). The sequence length between the SNP site and the reverse primer binding site is not critical.

STARP procedure and principle

The basic procedure of STARP includes competitive amplification of the two alleles followed by equal scaling amplification as a function of temperature. Competitive amplification is performed using the two AMAS-primers at the T a/e from 56 to 51 °C (Fig. 3a). At this T a/e range, the original complement of each allele is preferentially annealed by its corresponding AMAS-primer. The target primer-template duplex contains one artificial mismatch at the 3rd or 4th position from the 3′ terminus. This artificial mismatch does not significantly decrease the stability of the matched base pair at the 3′ terminus (Peyret et al. 1999), and it has a minor effect on PCR efficiency. Meanwhile, a small portion of the original complement of each allele may be annealed by its non-corresponding AMAS-primer. This non-target duplex contains two mismatches, one artificial and the other natural. The artificial mismatch further decreases the stability of the 3′ natural mismatch, and it dramatically reduces the extension efficiency of the non-target duplex. Therefore, AMAS-primer 1 and AMAS-primer 2 specifically amplify allele 1 and allele 2, respectively. The six bases (TATGAC-3′) at the 3′ region in tail 1 and tail 2 can randomly pair with the opposing bases in the template to form a T m balance region with same base-pair composition among the four duplexes in genotyping of each SNP locus. This T m balance region ensures that the actual T m values of the two AMAS-primers shift with similar scales. Thus, high amplification specificity is always achieved for both alleles.
Fig. 3

STARP procedure and principle. a Competitive amplification of the two alleles. The gray box indicates the T m balance region. The target duplexes (lower graphs) are efficiently extended (blue solid line), and the non-target duplexes (top graphs) may be extended but with very low efficiency. b Synthesis of complements using the common reverse primer. c Maintenance of high allele specificity. AMAS-primer 1 anneals to the newly generated allele 1 complement as matched template and is efficiently extended (right-top graph). Meanwhile, AMAS-primer 1 may anneal to the newly generated allele 2 complement, but is not extended because of three mismatches (in the ovals) at the 1st, 3rd, and 4th positions from the 3′ terminus in the duplex (right-lower graph). An analogous deduction is also applied to AMAS-primer 2 (right graphs). d Equal scaling amplification. The gray region indicates the same context of 3′ terminus, which ensures that PEA-primer 1 and PEA-primer 2 are always extended with similar efficiencies

Maintaining high allele specificity during STARP is another critical requirement. After specific amplification is initiated by the AMAS-primers, the common reverse primer anneals to both newly generated allele strands and synthesizes their complements that contain the priming sites of PEA-primers (Fig. 3b). These newly generated complements are amplified only by their corresponding AMAS-primers (Fig. 3c) as well as by their corresponding PEA-primers (Fig. 3d). Thus, high allele specificity is maintained during STARP. However, non-target annealing with AMAS-primers and the hairpin structure of PEA-primers will reduce the efficiency of amplification. These problems are addressed by appropriately increasing the T a/e for equal scaling amplification. At higher T a/e (62 °C), AMAS-primers no longer anneal to their original DNA templates because this T a/e is much higher than their actual T m values, and thus, the competitive amplification is terminated. Linearized PEA-primers replace AMAS-primers for proportional amplification. The ultimate PCR products of the two alleles carrying a single-stranded tail are discriminated by fluorescence signals or size separation.

Bi-allelic discrimination

Bi-allelic discrimination of STARP is achieved by fluorescence signals or size separation of the ultimate PCR products generated by the two PEA-primers. For the gel-free detection using fluorescence signals, PEA-primer 1 and PEA-primer 2 should be labeled with two different fluorophores at the 5′ phosphate group, respectively, and a quencher is attached to the thymidine at the 6th position from the 3′ terminus. The fluorophores must match the quencher and meet the optical requirements of a real-time PCR machine or fluorescence plate reader. In the present study, PEA-primer 1 and PEA-primer 2 were labeled with fluorophores FAM and HEX, respectively, and the quencher was Dabsyl. These dual-labeled PEA-primer 1 and PEA-primer 2 were designated as PEA1-QFAM and PEA2-QHEX, respectively (Fig. 1a; Table 1). The fluorescence intensity at the background level (\( {\text{F}}_{\text{FAM}}^{0} \) and \( {\text{F}}_{\text{HEX}}^{ 0} \)) and at the PCR endpoint (\( {\text{F}}_{\text{FAM}}^{1} \) and \( {\text{F}}_{\text{HEX}}^{1} \)) was measured in a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, laboratories Inc., Hercules, California, USA) at 33 °C. The \( {\text{F}}_{\text{FAM}}^{0} \) and \( {\text{F}}_{\text{HEX}}^{ 0} \) were pre-determined with ten replicas in the standard PCR mix with the exception of Taq. The average value of each fluorescence signal at the background level (\( {\bar{\text{F}}}_{\text{FAM}}^{0} \) and \( {\bar{\text{F}}}_{\text{HEX}}^{0} \)) was always used to calculate the fluorescence signal intensity from the actual PCR products (ΔFFAM = \( {\text{F}}_{\text{FAM}}^{ 1} - {\bar{\text{F}}}_{\text{FAM}}^{0} \) and ΔFHEX = \( {\text{F}}_{\text{HEX}}^{ 1} - {\bar{\text{F}}}_{\text{HEX}}^{0} \) in each SNP genotyping. Allelic discrimination was achieved by analyzing the ratio of ΔFFAM to ΔFHEX. The ratio values were clustered to form three distinct groups (homozygous allele 1, heterozygote, and homozygous allele 2) with a great gap among groups (Fig. 4).
Fig. 4

Validation of STARP for genotyping. Polyacrylamide gel (left) shows PCR fragments generated in AL 8/78, CIae 25, and their hybrid at each of nine STARP markers. Size separation was performed in an IR2 4300/4200 DNA Analyzer by denaturing PAGE. The names of all nine STARP markers (Table S3) and the determined genotypes (A1, H, and A2) were indicated on the gel. A1 and A2 present two homozygotes, and H presents heterozygote. Middle and right graphs indicate genotyping of an F2 population derived from the cross between AL 8/78 and CIae 25 with bi-allelic discrimination by fluorescence signals. The three clusters in each plot represent genotypes of homozygous allele 1, heterozygote, and homozygous allele 2

In addition to bi-allelic discrimination by fluorescence signals, the two PCR fragments also can be separated by regular polyacrylamide gel electrophoresis (PAGE) without labeling PEA-primers with dyes, and visualized after staining. For application in a DNA analyzer instead of staining, both PEA-primer 1 and PEA-primer 2 must be labeled with the same fluorophore at the 5′ phosphate group. The fluorophore must meet the optical requirements of the special DNA analyzer. In this study, both PEA-primer 1 and PEA-primer 2 were labeled with fluorophore IRDye® 700 and IRDye® 800, respectively (Table 1). Size separation of the PCR fragments was performed in an IR2 4300/4200 DNA Analyzer (LI-COR, Lincoln, NE, USA) by denaturing PAGE.

Optimization of STARP conditions

It is highly desirable to establish standard PCR conditions that are suitable for all genotyping assays, and are tolerant to variable DNA quality and quantity. To achieve high allele specificity, a high ratio of signal to noise and high amplification yield, PCR reactions need to be further optimized. First, the addition of 0.8 M betaine and 0.04% (W/V) bovine serum albumin (BSA) can reduce spurious amplification and increase the specificity of target sequences and amplification yield. Betaine and BSA also allow the PCR to overcome some low level of contaminants in the DNA samples. In addition, betaine plays a role in enlarging the optimal T a/e range and reducing the difference in PCR efficiencies between the two alleles. Second, the concentration of each PEA-primer should be equal to that of the common reverse primer and range from 150 to 250 nM, and the optimal ratios of PEA-primer to AMAS-primer range from 5:1 to 10:1. Third, the optimal DNA concentration varies from 10 to 100 ng per reaction in 10 µl total volume. We found that the DNA extraction methods did not significantly affect the allele specificity and amplification yield under the standard PCR conditions because both general methods (SDS and CTAB) and the commercial extraction kit (Qiagen, Valencia, CA, USA) showed similar genotyping results (data not shown).

Validation of STARP

This newly-developed STARP method was validated in rice, a common wheat (Triticum aestivum L.) progenitor Ae. tauschii, and sunflower, which represent DNA sources having a variety of genome complexes and natural PCR inhibitors. A total of 29 polymorphic loci (11 in rice, 15 in Ae. tauschii, and three in sunflower) involving three indels and all six types of SNPs (Table S3) were genotyped by STARP under the standard conditions. The PCR fragments of two homozygotes and their heterozygote in each locus were discriminated by size separation in an IR2 4300 DNA Analyzer using denaturing PAGE. Results showed that all STARP markers had high specificity with similar amplification efficiencies for the two alleles. Twelve STARP markers without spurious amplification in Ae. tauschii were further evaluated using one F2 population of 96 individuals derived from the cross between AL 8/78 and CIae 25. Results showed clear genotyping clusters (Fig. 4).

Discussion

The key target in the present study was to develop a novel SNP genotyping method that combines all the major advantages of a number of currently-used approaches in low operational costs, simple assay design, flexible throughputs, and high accuracy. During the development and optimization of the new method, we also focused on the compatibility of the technique with a variety of genotyping platforms. STARP attains all the requirements by using novel primer design in combination with unique PCR conditions.

Integrated advantages of STARP

Among the four major PCR-based approaches for genotyping of individual SNPs, the traditional allele-specific PCR is performed using two universal dual-labeled primers and two AS-primers in combination with a common reverse primer (Myakishev et al. 2001). Allele specificity is limited to the difference in the extension efficiencies of two duplexes between each AS-primer and complements of the two alleles. Five primers involved in a single tube behaves as multiplex PCR, in which one allele sequence is often preferentially amplified, resulting in the scarcity of the other allele sequence. The primer dimers and other spurious amplification products generated by dual-labeled primers also emit fluorescence, and thus cannot be distinguished from the PCR products of the target alleles. In addition, the hairpin sequence in dual-labeled primers is incorporated into PCR products. After denaturation during PCR, the newly-generated complement will form a hairpin structure and can be extended using the self-strand as a template, reducing amplification specificity and efficiency. Therefore, the allele-specific PCR method shows low SNP detection rate. The KASP (Kompetative Allele-Specific PCR) method, an improved allele-specific PCR developed by LGC (Middlesex, UK; http://www.lgcgroup.com), shows improved accuracy in allele discrimination and is commercially applied to SNP genotyping. However, the PCR reagents and AS-primer design highly rely on LGC, leading to higher operational costs and time-consumption on primer design. Bi-allelic discrimination in KASP is performed in real-time PCR machines or fluorescence plate readers that are not available in most laboratories. Furthermore, this method is sensitive to DNA quality and quantity.

In the present STARP method, these problems were addressed by application of the novel design of primers in combination with the optimal PCR conditions. The two PEA-primers were developed for common use in all genotyping assays to stringently target the two alleles with similar amplification efficiencies and for flexible detection. The application of the two dual-labeled PEA-primers for STARP assay significantly decreases the cost in PCR reagents (about 5 cents per 10 µl reaction) and labors. The capability of PEA-primers to stringently target the two alleles with similar amplification efficiencies dramatically increases SNP detection rate. The compatibility with multiple platforms is beneficial not only to global researchers to share genotyping conditions, but also to some laboratories to replace genotyping platforms in the future. Researchers design all the primers required for the SNP assays following the instruction described in this study, which saves researchers time and cost. The standard PCR conditions also make SNP genotyping easy to perform.

Principle of AMAS-primer design

The specificity of initial amplification for both alleles determines the genotyping accuracy. In traditional allele-specific PCR, each AS-primer anneals to complements of both alleles to form target and non-target duplexes with matched and mismatched base pair at its 3′ terminus, respectively. These two types of duplexes are extended with different efficiencies. Greater differences in the extension efficiency will result in higher allele specificity. AS-primer-template duplexes can form a total of 12 possible 3′ mismatches as follows: C•C, C•T, C•A, G•G, G•T, G•A, T•C, T•G, T•T, A•C, A•G, and A•A. In general, the 12 mismatches can be classified into three groups based on their extension efficiencies; groups I (C•C, G•G, G•A, A•G, A•A), II (C•T, T•C, T•T), and III (A•C, C•A, G•T, T•G) had significant, medium, and negligible effects on extension efficiency, respectively (Kwok et al. 1990; Huang et al. 1992; Stadhouders et al. 2010). To reduce the extension efficiency of these 3′ mismatched duplexes, one nucleotide in the AS-primer was substituted at the 2nd, 3rd, or 4th position from the 3′ terminus to form an artificial mismatch in its duplex (Newton et al. 1989; Cha et al. 1992). The observed position effect of the artificial mismatch on reducing extension efficiency is 2nd > 3rd > 4th. The 2nd mismatch could significantly reduce the extension efficiency of the 3′ matched duplex under certain conditions (Liu et al. 2012). Therefore, the 2nd position is excluded from nucleotide substitution in designing AMAS-primers of STARP. To achieve high-specific amplification for both alleles in STARP, the 3rd position for the nucleotide substitution is preferentially assigned to the AS-sequence that forms a 3′ mismatch of group III (G•T ≈ T•G > A•C ≈ C•A) in its non-target duplex and the 4th position is assigned to the AS-sequence that forms a 3′ mismatch of group I (C•C > G•G) in its non-target duplex. This asymmetric substitution forms three different nucleotides at the 1st, 3rd, and 4th positions from the 3′ terminus between the pair of AMAS-primers and greatly increases the genotyping accuracy of STARP.

The nucleotide substitution in AMAS-primer design must meet the following two critical requirements: (1) the target AMAS-primer-template duplex with one artificial mismatch should be extended with high efficiency similar to its perfectly matched duplex, and (2) the artificial mismatch should cause weaker thermal stabilization on the duplex. Because the internal mismatch and the 3′ terminal mismatch have similar trends in PCR efficiency (Stadhouders et al. 2010), nucleotide C can be substituted with G, T, or A, and their corresponding duplexes will contain a G•G, T•G, or A•G mismatch, respectively. Out of these three mismatches, G•G and A•G (group I) significantly reduces the PCR efficiency. Therefore, in AMAS-primer design for STARP, nucleotide C is substituted with T to form a mismatch of group III (T•G). For the same reason, nucleotide T is substituted with C. Nucleotide G can be substituted with C, T, or A, and their corresponding duplexes will contain a C•C, T•C, or A•C mismatch, respectively. Out of these three mismatches, C•C significantly reduces the PCR efficiency. Both T•C and A•C have minor effects on the extension efficiency, but A•C causes weaker stabilization on the duplex than T•C (Peyret et al. 1999). Therefore, nucleotide G is substituted with A. Finally, A is substituted with C due to the weakest stabilization on the duplex. Therefore, the nucleotide substitution in AMAS-primer design should follow this principle: A → C, T → C, G → A, and C → T.

The 3′ termini in AMAS-primers are often extended with different efficiencies (C ≥ G ≥ T > A) (Ayyadevara et al. 2000). This difference can be rectified by assigning a tail. Since the amplification efficiency of PEA-primer 1 is a little lower than that of PEA-primer 2, Tail 1 is preferentially assigned to SNP allele C and then G, and tail 2 is assigned to A and then T. Thus, This AMAS-primer design approach ensures that STARP always amplifies the two alleles with high allele specificity and similar efficiencies under the unique conditions.

Standardization of PCR conditions

It is desirable to perform STARP for all genotyping assays under standard PCR conditions. To achieve the requirement, we first explored the correlation between the predicted T m value of AS-sequence and the optimal T a/e range of its corresponding AMAS-primer. Three DNA samples (two homozygotes and their heterozygote) in sunflower were used to examine 66 AMAS-primers (33 SNP loci) for their optimal T a/e ranges by thermal gradient PCR with T a/e ranging from 40 to 62 °C (Table S2). The observed optimal T a/e ranges varied from 7 (SFW02101F2) to 22 °C (36 AMAS-primers) with an average T a/e range of 18.7 °C. Out of these T a/e ranges obtained from the 66 AMAS-primers, the subtracted value between the predicted T m value and its minimum optimal T a/e varied from 2 (SFW02249BF2 and SFW00829F1) to 23 °C (SFW03126F1) with an average subtracted value of 14 °C. The subtracted value between the predicted T m value and its maximum optimal T a/e varied from −1 (5 AS-primers) to 12 °C (SFW00232F1) with an average subtracted value of 5 °C. This correlation was used to assist with potentially developing a universal PCR program in STARP for high specificity with similar amplification efficiencies for the two alleles. In addition, we found that most spurious amplification disappeared when the T a/e was higher than 50 °C (data not shown). After carefully considering the obtained results, we designed the AS-sequences having T m values ranging from 54 to 58 °C and performed competitive amplification at T a/e from 56 to 51 °C.

The results from this study showed that STARP clearly discriminated between the two alleles of all SNPs and indels. STARP makes extensive use of genotyping more feasible because of its integrated advantages and high compatibility with various genotyping platforms. Vast variations in DNA sequences are being unearthed by many genome sequencing projects and association mapping by genotyping by sequencing (GBS) that are occurring globally. Therefore, this method has great potential for broad exploitation of SNPs and indels across all biological organisms in agriculture, medicine, and forensics.

Author contribution statement

YML and LLQ conceived and designed the experiments, performed the experiments, and analyzed data. WSC helped with real-time PCR. GJM validated this method. SSX contributed to the materials and method validation. YML wrote the paper. WSC, LLQ, SSX, and GJM commented on the manuscript before submission

Notes

Acknowledgements

We thank Drs. Yiqun Weng and Zengcui Zhang for critically reviewing this manuscript. This research was supported by the USDA-ARS National Sclerotinia Initiative, Grant No. 5442-21220-028-00D and the USDA-ARS CRIS Project No. 3060-21000-039-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments were performed in compliance with the current laws of the USA.

Supplementary material

122_2016_2838_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 15 kb)
122_2016_2838_MOESM2_ESM.xlsx (53 kb)
Supplementary material 2 (XLSX 52 kb)
122_2016_2838_MOESM3_ESM.docx (45 kb)
Supplementary material 3 (DOCX 44 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Authors and Affiliations

  • Y. M. Long
    • 1
  • W. S. Chao
    • 2
  • G. J. Ma
    • 3
  • S. S. Xu
    • 2
  • L. L. Qi
    • 2
    Email author
  1. 1.Department of Plant SciencesNorth Dakota State UniversityFargoUSA
  2. 2.USDA-Agricultural Research Service, Northern Crop Science LaboratoryFargoUSA
  3. 3.Department of Plant PathologyNorth Dakota State UniversityFargoUSA

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