AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a "true" single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0 microL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor-saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.
AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a "true" single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0 microL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor-saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.
Authors: Axel zur Hausen; J Bart A Crusius; Laura S Murillo; Behrooz Z Alizadeh; Servaas A Morré; Chris J L M Meijer; Adriaan J C van den Brule; Amado Salvador Peña Journal: Int J Cancer Date: 2003-12-10 Impact factor: 7.396
Authors: R Sachidanandam; D Weissman; S C Schmidt; J M Kakol; L D Stein; G Marth; S Sherry; J C Mullikin; B J Mortimore; D L Willey; S E Hunt; C G Cole; P C Coggill; C M Rice; Z Ning; J Rogers; D R Bentley; P Y Kwok; E R Mardis; R T Yeh; B Schultz; L Cook; R Davenport; M Dante; L Fulton; L Hillier; R H Waterston; J D McPherson; B Gilman; S Schaffner; W J Van Etten; D Reich; J Higgins; M J Daly; B Blumenstiel; J Baldwin; N Stange-Thomann; M C Zody; L Linton; E S Lander; D Altshuler Journal: Nature Date: 2001-02-15 Impact factor: 49.962
Authors: J D McPherson; M Marra; L Hillier; R H Waterston; A Chinwalla; J Wallis; M Sekhon; K Wylie; E R Mardis; R K Wilson; R Fulton; T A Kucaba; C Wagner-McPherson; W B Barbazuk; S G Gregory; S J Humphray; L French; R S Evans; G Bethel; A Whittaker; J L Holden; O T McCann; A Dunham; C Soderlund; C E Scott; D R Bentley; G Schuler; H C Chen; W Jang; E D Green; J R Idol; V V Maduro; K T Montgomery; E Lee; A Miller; S Emerling; R Gibbs; S Scherer; J H Gorrell; E Sodergren; K Clerc-Blankenburg; P Tabor; S Naylor; D Garcia; P J de Jong; J J Catanese; N Nowak; K Osoegawa; S Qin; L Rowen; A Madan; M Dors; L Hood; B Trask; C Friedman; H Massa; V G Cheung; I R Kirsch; T Reid; R Yonescu; J Weissenbach; T Bruls; R Heilig; E Branscomb; A Olsen; N Doggett; J F Cheng; T Hawkins; R M Myers; J Shang; L Ramirez; J Schmutz; O Velasquez; K Dixon; N E Stone; D R Cox; D Haussler; W J Kent; T Furey; S Rogic; S Kennedy; S Jones; A Rosenthal; G Wen; M Schilhabel; G Gloeckner; G Nyakatura; R Siebert; B Schlegelberger; J Korenberg; X N Chen; A Fujiyama; M Hattori; A Toyoda; T Yada; H S Park; Y Sakaki; N Shimizu; S Asakawa; K Kawasaki; T Sasaki; A Shintani; A Shimizu; K Shibuya; J Kudoh; S Minoshima; J Ramser; P Seranski; C Hoff; A Poustka; R Reinhardt; H Lehrach Journal: Nature Date: 2001-02-15 Impact factor: 49.962
Authors: Richard Novak; Yong Zeng; Joe Shuga; Gautham Venugopalan; Daniel A Fletcher; Martyn T Smith; Richard A Mathies Journal: Angew Chem Int Ed Engl Date: 2011-01-10 Impact factor: 15.336