Simultaneous Genotyping of Single-nucleotide Polymorphisms in Alcoholism-related Genes Using Duplex and Triplex Allele-specific PCR with Two-step Thermal Cycles

Shirasu, Naoto; Kuroki, Masahide

doi:10.2116/analsci.30.1093

Cited by 6 publications

(12 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Table 1 summarizes the results of the genotyping by the duplex PCR-RFLP in a single-tube reaction and the previous sequencing analysis. The newly determined genotypes completely matched the previous sequencing results, 10 suggesting that our single-tube PCR-RFLP method is highly accurate, and may be practically applicable in genetic diagnosis.…”

Section: Resultssupporting

confidence: 69%

“…Gene fragments containing each allele of ADH1B (ADH1B*1 or ADH1B*2) and ALDH2 (ALDH2*1 or ALDH2*2) were custom synthesized and cloned into the pIDTSMART-Amp vector (Integrated DNA Technologies, Coralville, IA, USA). 10 FastDigest MslI and TscAI were purchased from Thermo Scientific. Agarose S was purchased from Nippon Gene (Toyama, Japan).…”

Section: Reagents and Subjectsmentioning

confidence: 99%

“…10 Briefly, 18 μl of 50 mM NaOH was added to a microcentrifuge tube containing a human hair root (approximately 1 cm in length). The sample was incubated at 95 C for 10 min, and then 2 μl of 1 M Tris-HCl (pH 8.0) was added.…”

Section: Template Preparationmentioning

confidence: 99%

“…7,8 However, these methods are generally time-consuming, and require specialized probes and expensive instruments. The allele-specific PCR is a relatively simple and cost-effective method compared with other genotyping techniques, 9,10 however, multiple reaction parameters must be optimized for effective allele-specific amplification, which is often labor-intensive. 11 Moreover, the allelic specificity is easily affected, and sometimes lost by subtle changes of the reaction conditions.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes

Shirasu

Yasunaga

2016

ANAL. SCI.

Self Cite

View full text Add to dashboard Cite

Single nucleotide polymorphisms (SNPs) in ADH1B and ALDH2, which encode alcohol dehydrogenase 1B and aldehyde dehydrogenase 2, respectively, are responsible for ethanol-metabolizing activity and alcohol-related disease. We developed a simple and rapid polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method for the simultaneous genotyping of the genes in a single-tube reaction. Using an alkaline lysate of a human hair root as a template, SNP-containing regions of each gene were PCR-amplified simultaneously. The PCR products were directly applied to double restriction digestion, followed by agarose gel band analysis. The duplex PCR-RFLP allows for the simultaneous and accurate determination of the SNP genotypes of ADH1B and ALDH2 within 1.2 h.

show abstract

Section: Resultssupporting

confidence: 69%

Section: Reagents and Subjectsmentioning

confidence: 99%

Section: Template Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes

Shirasu

Yasunaga

2016

ANAL. SCI.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mammalian cells have weak cell membranes but do not have cell walls, whereas bacteria possess both10; thus, the chromosomal DNA of mammalian cells can easily be eluted from cells11. To remove the time- and money-intensive DNA isolation step, real-time PCR with crude DNA extraction (intermediate direct qPCR) through boiling has been performed with clinical samples, including hair roots.…”

mentioning

confidence: 99%

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells

Soejima

Xiao

Abe

2016

Sci Rep

View full text Add to dashboard Cite

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

show abstract

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism‐related genes using PCR‐based assays to understand the link between an individual's genotype and phenotype

Shirasu,

Yasunaga

2023

Biochem Molecular Bio Educ

Self Cite

View full text Add to dashboard Cite

Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele‐specific (AS) PCR and a PCR‐restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2‐step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS‐PCR, the target regions of the G‐ or A‐alleles of both genes are allele‐specifically amplified in a single PCR tube. In the PCR‐RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double‐digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.

show abstract

Simultaneous Genotyping of Single-nucleotide Polymorphisms in Alcoholism-related Genes Using Duplex and Triplex Allele-specific PCR with Two-step Thermal Cycles

Cited by 6 publications

References 11 publications

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism‐related genes using PCR‐based assays to understand the link between an individual's genotype and phenotype

Contact Info

Product

Resources

About