pcr-based snp genotyping assay development for animal … · pcr-based snp genotyping assay...

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PCR-Based SNP Genotyping

Assay Development

for Animal and Plant Genomic

Research

Cassie Keppel and Luke Linz, Douglas Scientific, Alexandria, MN, USA

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Assay design

Master Mix Supplier

KASP™1536 V4.0 Master Mix LGC Genomics

TaqMan® GTXpress™ Master Mix Life Technologies

TaqMan® Genotyping Master Mix Life Technologies

PerfeCTa® qPCR ToughMix® Quanta BioSciences

SensiFAST™ Lo-ROX Genotyping Kit Bioline

SsoAdvanced™ Universal Probes Supermix Bio-Rad Laboratories

Six different commercially available master mixes were tested for their ability to successfully

genotype three genes; CAPN1-4751, PMCH and UoGCAST from purified bovine DNA


Results: CAPN1-4751 (Class 1 SNP)

Master Mix Comparison for Bovine SNP CAPN1-4751: Six different commercially available master mixes were used for SNP genotyping with purified bovine DNA.

The SensiFAST Genotyping Kit delivered an improved performance versus the TaqMan

Genotyping Kit, successfully discriminated all twenty nine of the target SNPs


Results: UoGCAST (Class 3 SNP)

Master Mix Comparison for Bovine SNP UoGCAST: Six different commercially available master mixes were used for SNP genotyping with purified bovine DNA.


GTXpress Kit, successfully discriminated all twenty nine of the target SNPs


Results: PMCH (Class 4 SNP)

Master Mix Comparison for Bovine SNP PMCH: Six different commercially available master mixes were used for SNP genotyping with purified bovine DNA.


GTXpress Kit, successfully discriminated all twenty nine of the target SNPs


*Samples did not fall within a cluster

SNP Genotyping Call Results

SNP

KASP

1536

V4.0

TaqMan

GTXpress

TaqMan

Genotyping

PerfeCTa

ToughMix

SensiFAST

Genotyping

Bio-Rad

SsoAdvanced

Leptin

(CC/CT/TT)2/19/8 2/19/8 2/19/8 2/19/8 2/19/8 2/19/8

CAPN1-4751

(CC/CT/TT)8/10/11 8/10/11 8/9*/11 8/10/11 8/10/11 8/10/11

PMCH

(AA/AT/TT)13/14/2 13/13*/2 13/14/2 13/14/2 13/14/2 13/14/2

UoGCAST

(CC/CG/GG)8/10/11 8/10*/10 8/10/11 8/10/11 8/10/11 8/10/11

The SensiFAST Genotyping, KASP, PerfeCTa and SsoAdvanced kits were able to discriminate

all twenty nine of the SNPs and cluster them with a greater than 97% call rating, even with

class 4 SNPs (A/T substitution). The TaqMan kits that failed to call some genotypes


Conclusion

All SNP genotyping reactions in this experiment produced clusters that

were easily scored in IntelliScore™ data analysis software. Three samples,

from the TaqMan GTXpress and TaqMan Genotyping kits were not scored

because of poor amplification.

There was no apparent advantage in using the KASP system as it did not

give cleaner or clearer clusters or scoring than the TaqMan based kits.

This study demonstrates that the SensiFAST™ Genotyping Kit can be

used on the Array Tape Platform, with bovine samples in miniaturized

reaction volumes, to produce easily scored SNP genotyping results, that

are repeatable, reliability and accuracy, even with class 4 SNPs.

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Thank you……

…for your attention

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SensiFAST Genotyping Kit

A combination of the latest advances in buffer chemistry, together with high-affinity

antibodies for the hot-start DNA polymerase system ensures that the SensiFAST™

Genotyping Kit produces highly-specific, ultra-sensitive real-time PCR with clear

allelic discrimination and outstanding allele clustering. The SensiFAST™ Genotyping

Kit is ideal for high through-put with many dual-labeled probe assays (including

TaqMan® probe based assays) from a wide range of sources, including human,

animal and plant samples.

The advanced buffer chemistry in the SensiFAST™ Genotyping Kit provides higher

stringency and more specific binding of the allele-specific probes, resulting in much

narrower probe melting temperature windows, leading to a wider and clearer

separation of allele clusters. The kit allows robust genotype calling enabling

reproducible and accurate results. The advanced buffer also means that minimal

optimization is required to achieve fast, reproducible and accurate results, ideal for

high throughput screening.

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Kits

Product Size Cat No.

SensiFAST™ Genotyping Kits

SensiFAST™ Hi-ROX Genotyping Kit 200 x 20µl BIO-35002



SensiFAST™ Lo-ROX Genotyping Kit 200 x 20µl BIO-34002



SensiFAST™ HRM Kit 200 x 20µl BIO-32002



SensiFAST™ SYBR and Probe Kits

SensiFAST™ SYBR Hi-ROX Kit 200 x 20µl BIO-92002



SensiFAST™ SYBR Lo-ROX Kit 200 x 20µl BIO-94002



SensiFAST™ SYBR No-ROX Kit 200 x 20µl BIO-98002



SensiFAST™ SYBR & Fluorescein Kit 200 x 20µl BIO-96002



SensiFAST™ Probe Hi-ROX Kit 200 x 20µl BIO-82002



SensiFAST™ Probe Lo-ROX Kit 200 x 20µl BIO-84002



SensiFAST™ Probe No-ROX Kit 200 x 20µl BIO-86002



SensiFAST™ cDNA Synthesis Kits

SensiFAST™ cDNA Synthesis Kit 50 reactions BIO-65053

SensiFAST™ cDNA Synthesis Kit 250 reactions BIO-65054

Product Size Cat No.

SensiFAST™ SYBR and Probe One-Step Kits

SensiFAST™ SYBR Hi-ROX One-Step Kit 100 x 20µl BIO-73001

SensiFAST™ SYBR Hi-ROX One-Step Kit 500 x 20µl BIO-73005

SensiFAST™ SYBR Lo-ROX One-Step Kit 100 x 20µl BIO-74001

SensiFAST™ SYBR Lo-ROX One-Step Kit 500 x 20µl BIO-74005

SensiFAST™ SYBR No-ROX One-Step Kit 100 x 20µl BIO-72001

SensiFAST™ SYBR No-ROX One-Step Kit 500 x 20µl BIO-72005

SensiFAST™ SYBR & Fluorescein One-Step Kit 100 x 20µl BIO-75001

SensiFAST™ SYBR & Fluorescein One-Step Kit 500 x 20µl BIO-75005

SensiFAST™ Probe Hi-ROX One-Step Kit 100 x 20µl BIO-77001

SensiFAST™ Probe Hi-ROX One-Step Kit 500 x 20µl BIO-77005

SensiFAST™ Probe Lo-ROX One-Step Kit 100 x 20µl BIO-78001

SensiFAST™ Probe Lo-ROX One-Step Kit 500 x 20µl BIO-78005

SensiFAST™ Probe No-ROX One-Step Kit 100 x 20µl BIO-76001

SensiFAST™ Probe No-ROX One-Step Kit 500 x 20µl BIO-76005


Materials and Methods

SensiFAST Genotyping Lo-ROX was compared to several commercially available

master mixes using maize seeds and bovine tissue along with allele-specific

hydrolysis probes

Purified genomic DNA and crude sample preparation methods were used for both

bovine samples and the assays were run on the Array Tape® Platform

from Douglas Scientific.

DNA was purified from twenty nine bovine samples using a either DNeasy Blood

and Tissue Kit or NaOH (by heating minced tissue in sodium hydroxide solution,

neutralized with Tris buffer).


Assay design

There are two competing technologies for high-throughput real-time PCR analysis of SNPs. Customers can

either use probes (A) or primers with unique tail sequences (KASP) (B). KASP is more robustness, but

probes give higher sensitivity and specificity.

pcr-based snp genotyping assay development for animal … · pcr-based snp genotyping assay...

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