GenotypingPortal3

Genotyping Portal

A comprehensive (and freely available) online resource about methods for DNA genotyping, screening and sequencing

PCR techniques

PCR (Polymerase Chain Reaction): Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487-91.

M-PCR (Multiplex PCR): Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1988 Dec 9;16(23):11141-56.

N-PCR (Nested PCR): Haqqi TM, Sarkar G, David CS, Sommer SS. Specific amplification with PCR of a refractory segment of genomic DNA. Nucleic Acids Res. 1988 Dec 23;16(24):11844.

TD-PCR (Touch-Down PCR: Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008.

TU-PCR (Touch-Up PCR): Rowther FB, Kardooni H, Warr T. TOUCH-UP gradient amplification method. J Biomol Tech. 2012 Apr;23(1):1-3.

SD-PCR (SlowDown PCR): Bachmann HS, Siffert W, Frey UH. Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique. Pharmacogenetics. 2003 Dec;13(12):759-66.

S-PCR (Subcycling PCR): Liu Q, Sommer SS. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene. Biotechniques. 1998 Dec;25(6):1022-8.

IS-PCR (In Situ PCR): Haase AT, Retzel EF, Staskus KA. Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4971-5.

HS-PCR (Hot Start PCR): Chou Q, Russell M, Birch DE, Raymond J, Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. 1992 Apr 11;20(7):1717-23.

COLD-PCR (CO-amplification at Lower Denaturation temperature-PCR): Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med. 2008 May;14(5):579-84.

ice-COLD-PCR (Improved and Complete Enrichment-COLD-PCR): Milbury CA, Li J, Makrigiorgos GM. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Res. 2011 Jan;39(1):e2.

RAPD (Random Amplified Polymorphic DNA): Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531-5.

AP-PCR (Arbitrarily Primed PCR): Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990 Dec 25;18(24):7213-8.

d-PCR (Digital PCR): Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41.

LAMP (Loop-mediated isothermal amplification): Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000 Jun 15;28 (12):E63.

NASBA (Nucleic Acid Sequence-Based Amplification): Compton J. Nucleic acid sequence-based amplification. Nature. 1991 Mar 7;350(6313):91-2.

HDA (Helicase-Dependent Amplification): Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004 Aug;5(8):795-800.

ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids): Uemori T, Mukai H, Takeda O, Moriyama M, Sato Y, Hokazono S, Takatsu N, Asada K, Kato I. Investigation of the molecular mechanism of ICAN, a novel gene amplification method. J Biochem. 2007 Aug;142(2):283-92.

RCA (Rolling Circle Amplification): Fire A, Xu SQ. Rolling replication of short DNA circles. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4641-5.

RPA (Recombinase Polymerase Amplification): Piepenburg O, Williams CH, Stemple DL, Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006 Jul;4(7):e204.

SDA (Strand Displacement Amplification): Walker GT, Fraiser MS, Schram JL, Little MC, Nadeau JG, Malinowski DP. Strand displacement amplification--an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 1992 Apr 11;20(7):1691-6.

Smartphone-based convective PCR: Rajendran VK, Bakthavathsalam P, Bergquist PL, Sunna A. A portable nucleic acid detection system using natural convection combined with a smartphone. Biosens Bioelectron. 2019 Jun 1;134:68-75.

cPCR (Convective PCR): Zhang S, Wang J, Zhuo Z, Su X, Chen M, Chen W, Li T, Zhang D, Min X, Ge S, Xia N. An efficient isothermal PCR method for on-site detection of nucleic acid. Biotechniques. 2019 Aug;67(2):63-69.

SMAP 2 (Smart Amplification Process version 2): Mitani Y, Lezhava A, Kawai Y, Kikuchi T, Oguchi-Katayama A, Kogo Y, Itoh M, Miyagi T, Takakura H, Hoshi K, Kato C, Arakawa T, Shibata K, Fukui K, Masui R, Kuramitsu S, Kiyotani K, Chalk A, Tsunekawa K, Murakami M, Kamataki T, Oka T, Shimada H, Cizdziel PE, Hayashizaki Y. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nat Methods. 2007 Mar;4(3):257-62.

SPIA (Single Primer Isothermal Amplification): Kurn N, Chen P, Heath JD, Kopf-Sill A, Stephens KM, Wang S. Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications. Clin Chem. 2005 Oct;51(10):1973-81.

HDA-TaqMan: Tong Y, Tang W, Kim HJ, Pan X, Ranalli T, Kong H. Development of isothermal TaqMan assays for detection of biothreat organisms. Biotechniques. 2008 Nov;45(5):543-57.

Melting Curve Analysis: Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques. 1997 Jan;22(1):130-1, 134-8.

Ligase detection reaction: Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):189-93.

Multiplex-LAMP: Tanner NA, Zhang Y, Evans TC Jr. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification. Biotechniques. 2012 Aug;53(2):81-9. http://www.ncbi.nlm.nih.gov/pubmed/23030060

MegaPlex PCR: Meuzelaar LS, Lancaster O, Pasche JP, Kopal G, Brookes AJ. MegaPlex PCR: a strategy for multiplex amplification. Nat Methods. 2007 Oct;4(10):835-7.

TAIL-PCR (Thermal Asymmetric InterLaced PCR): Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques. 2007 Nov;43(5):649-50.

RAPD-HRM: Cambier S, Gonzalez P, Durrieu G, Bourdineaud JP. Cadmium-induced genotoxicity in zebrafish at environmentally relevant doses. Ecotoxicol Environ Saf. 2010 Mar;73(3):312-9.

AFLP (Amplified fragment length polymorphism): AFLP: a new technique for DNA fingerprinting. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. Nucleic Acids Res. 1995 Nov 11;23(21):4407-14.

Antisense PCR: Brisco MJ, Bartley PA, Morley AA. Antisense PCR: A simple and robust method for performing nested single-tube PCR. Anal Biochem. 2011 Feb 15;409(2):176-82.

Quasi-digital PCR: Sundberg SO, Wittwer CT, Zhou L, Palais R, Dwight Z, Gale BK. Quasi-digital PCR: Enrichment and quantification of rare DNA variants. Biomed Microdevices. 2014 Aug;16(4):639-44.

Primer-dimers: Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997 Aug 15;25(16):3235-41.

Multiplex PCR design: Broude NE, Zhang L, Woodward K, Englert D, Cantor CR. Multiplex allele-specific target amplification based on PCR suppression. Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):206-11.

qPCR standards: Viljoen CD, Thompson GG, Sreenivasan S. Stability of ultramer as copy number standards in real-time PCR. Gene. 2013 Mar 1;516(1):143-5.

Use of BSA: Kreader CA. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol. 1996 Mar;62(3):1102-6.

Use of Betaine in PCR: Henke W, Herdel K, Jung K, Schnorr D, Loening SA. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 1997 Oct 1;25(19):3957-8.

Use of DMSO in PCR: Winship PR. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 1989 Feb 11;17(3):1266.

Use of 1,2-propanediol-trehalose mixture in PCR: Horáková H, Polakovičová I, Shaik GM, Eitler J, Bugajev V, Dráberová L, Dráber P. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer. BMC Biotechnol. 2011 Apr 18;11:41.

Use of EG and PG in PCR: Zhang Z, Yang X, Meng L, Liu F, Shen C, Yang W. Enhanced amplification of GC-rich DNA with two organic reagents. Biotechniques. 2009 Sep;47(3):775-9.

Comparison of PCR additives: Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I. Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn. 2006 Nov;8(5):544-50.

Combinations of PCR additives: Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006 Sep 1;347(3):747-51.

Flurorescent labeling of PCR products: Schuelke M. An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol. 2000 Feb;18(2):233-4.

Flurorescent labeling of PCR products: Neilan BA, Wilton AN, Jacobs D. A universal procedure for primer labelling of amplicons. Nucleic Acids Res. 1997 Jul 15;25(14):2938-9.

EvaGreen: Mao F, Leung WY, Xin X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol. 2007 Nov 9;7:76.

qPCR dyes: Eischeid AC. SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR. BMC Res Notes. 2011 Jul 28;4:263.

qPCR dyes: Gudnason H, Dufva M, Bang DD, Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 2007;35(19):e127.

qPCR dyes: Oscorbin IP, Belousova EA, Zakabunin AI, Boyarskikh UA, Filipenko ML. Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP). Biotechniques. 2016 Jul 1;61(1):20-5.

qPCR dyes: Haines AM, Tobe SS, Linacre A. Optimization of Diamond Nucleic Acid Dye for quantitative PCR. Biotechniques. 2016 Oct 1;61(4):183-189.

qPCR master mixes: Yang J, Kemps-Mols B, Spruyt-Gerritse M, Anholts J, Claas F, Eikmans M. The source of SYBR green master mix determines outcome of nucleic acid amplification reactions. BMC Res Notes. 2016 Jun 4;9:292.

qPCR normalization: Marullo M, Zuccato C, Mariotti C, Lahiri N, Tabrizi SJ, Di Donato S, Cattaneo E. Expressed Alu repeats as a novel, reliable tool for normalization of real-time quantitative RT-PCR data. Genome Biol. 2010 Jan 28;11(1):R9.

qPCR normalization: Rihani A, Van Maerken T, Pattyn F, Van Peer G, Beckers A, De Brouwer S, Kumps C, Mets E, Van der Meulen J, Rondou P, Leonelli C, Mestdagh P, Speleman F, Vandesompele J. Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments. PLoS One. 2013 Aug 14;8(8):e71776.

qPCR normalization: Hernandez-Segura A, Rubingh R, Demaria M. Identification of stable senescence-associated reference genes. Aging Cell. 2019 Apr;18(2):e12911.

qPCR normalization: Vanhauwaert S, Van Peer G, Rihani A, Janssens E, Rondou P, Lefever S, De Paepe A, Coucke PJ, Speleman F, Vandesompele J, Willaert A. Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies. PLoS One. 2014 Oct 13;9(10):e109091.

Colorimetric detection: Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. 2009 Mar;46(3):167-72.

Colorimetric detection: Tanner NA, Zhang Y, Evans TC Jr. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 2015 Feb 1;58(2):59-68.

Colorimetric detection: Miyamoto S, Sano S, Takahashi K, Jikihara T. Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes. Anal Biochem. 2015 Mar 15;473:28-33.

Comparison of thermal cyclers: Kim YH, Yang I, Bae YS, Park SR. Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Biotechniques. 2008 Apr;44(4):495-505.

Comparison of thermal cyclers: Schoder D, Schmalwieser A, Schauberger G, Hoorfar J, Kuhn M, Wagner M. Novel approach for assessing performance of PCR cyclers used for diagnostic testing. J Clin Microbiol. 2005 Jun;43(6):2724-8.

Comparison of HRM instruments: Li M, Zhou L, Palais RA, Wittwer CT. Genotyping accuracy of high-resolution DNA melting instruments. Clin Chem. 2014 Jun;60(6):864-72.

Comparison of HRM instruments: Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem. 2006 Mar;52(3):494-503.

PCR Optimization: Roux KH. Optimization and troubleshooting in PCR. Cold Spring Harb Protoc. 2009 Apr;2009(4):pdb.ip66.

PCR success: Benita Y, Oosting RS, Lok MC, Wise MJ, Humphery-Smith I. Regionalized GC content of template DNA as a predictor of PCR success. Nucleic Acids Res. 2003 Aug 15;31(16):e99.

PCR failure: Andreson R, Möls T, Remm M. Predicting failure rate of PCR in large genomes. Nucleic Acids Res. 2008 Jun;36(11):e66.

PCR Contamination: Scherczinger CA, Ladd C, Bourke MT, Adamowicz MS, Johannes PM, Scherczinger R, Beesley T, Lee HC. A systematic analysis of PCR contamination. J Forensic Sci. 1999 Sep;44(5):1042-5.

PCR Contamination: Urban C, Gruber F, Kundi M, Falkner FG, Dorner F, Hämmerle T. A systematic and quantitative analysis of PCR template contamination. J Forensic Sci. 2000 Nov;45(6):1307-11.

Software for qPCR analysis: Krähenbühl S, Studer F, Guirou E, Deal A, Mächler P, Hosch S, Mpina M, Mswata S, Daubenberger C, Schindler T. ELIMU-MDx: a web-based, open-source platform for storage, management and analysis of diagnostic qPCR data. Biotechniques. 2020 Jan;68(1):22-27.

Software for qPCR analysis: Rancurel C, van Tran T, Elie C, Hilliou F. SATQPCR: Website for statistical analysis of real-time quantitative PCR data. Mol Cell Probes. 2019 Aug;46:101418.

DNA Extraction

DNA extraction-Salting out: Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215.

Salting out: Lahiri DK, Nurnberger JI Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res. 1991 Oct 11;19(19):5444.

DNA-Buccal cells: Richards B, Skoletsky J, Shuber AP, Balfour R, Stern RC, Dorkin HL, Parad RB, Witt D, Klinger KW. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet. 1993 Feb;2(2):159-63.

FFPE-DNA Extraction: Goelz SE, Hamilton SR, Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun. 1985 Jul 16;130(1):118-26.

PEP (Primer-Extension Preamplification)-WGA: Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847-51.

MDA (Multiple Displacement Amplification)-GWA: Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J, Driscoll M, Song W, Kingsmore SF, Egholm M, Lasken RS. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5261-6.

WGA-Exome: Indap AR, Cole R, Runge CL, Marth GT, Olivier M. Variant discovery in targeted resequencing using whole genome amplified DNA. BMC Genomics. 2013 Jul 10;14:468.

WGA-Reliability: Berthier-Schaad Y, Kao WH, Coresh J, Zhang L, Ingersoll RG, Stephens R, Smith MW. Reliability of high-throughput genotyping of whole genome amplified DNA in SNP genotyping studies. Electrophoresis. 2007 Aug;28(16):2812-7.

Pooled whole blood: Craig JE, Hewitt AW, McMellon AE, ..., Visscher PM, Montgomery GW, MacGregor S. Rapid inexpensive genome-wide association using pooled whole blood. Genome Res. 2009 Nov;19(11):2075-80.

Pooled DNA: Arnheim N, Strange C, Erlich H. Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6970-4.

Use of FTA cards: Salvador JM, De Ungria MC. Isolation of DNA from saliva of betel quid chewers using treated cards. J Forensic Sci. 2003 Jul;48(4):794-7.

DNA-Blood Storage: Richardson AJ, Narendran N, Guymer RH, Vu H, Baird PN. Blood storage at 4 degrees C-factors involved in DNA yield and quality. J Lab Clin Med. 2006 Jun;147(6):290-4

Nanodrop-DNA quantification: Desjardins P, Conklin D. NanoDrop microvolume quantitation of nucleic acids. J Vis Exp. 2010 Nov 22;(45). doi:pii: 2565. 10.3791/2565.

PicoGreen-DNA quantification: Ahn SJ, Costa J, Emanuel JR. PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res. 1996 Jul 1;24(13):2623-5.

Dyes for Gel Electrophoresis: Haines AM, Tobe SS, Kobus HJ, Linacre A. Properties of nucleic acid staining dyes used in gel electrophoresis. Electrophoresis. 2015 Mar;36(6):941-4.

qPCR-Quantification: Nicklas JA, Buel E. Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples. J Forensic Sci. 2003 Sep;48(5):936-44.

RNA integrity: Brisco MJ, Morley AA. Quantification of RNA integrity and its use for measurement of transcript number. Nucleic Acids Res. 2012 Oct;40(18):e144.

DNA quantification-Dyes: Bruijns BB, Tiggelaar RM, Gardeniers JG. Fluorescent cyanine dyes for the quantification of low amounts of dsDNA. Anal Biochem. 2016 Oct 15;511:74-9.

Sample-to-result platform: Lafleur LK, Bishop JD, Heiniger EK, Gallagher RP, Wheeler MD, Kauffman P, Zhang X, Kline EC, Buser JR, Kumar S, Byrnes SA, Vermeulen NM, Scarr NK, Belousov Y, Mahoney W, Toley BJ, Ladd PD, Lutz BR, Yager P. A rapid, instrument-free, sample-to-result nucleic acid amplification test. Lab Chip. 2016 Oct 7;16(19):3777-87.

Sample-to-result platform: Tang R, Yang H, Gong Y, You M, Liu Z, Choi JR, Wen T, Qu Z, Mei Q, Xu F. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection. Lab Chip. 2017 Mar 29;17(7):1270-1279.

Blood and Buccal DNA-Comparison: Hansen TV, Simonsen MK, Nielsen FC, Hundrup YA. Collection of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort: comparison of the response rate and quality of genomic DNA. Cancer Epidemiol Biomarkers Prev. 2007 Oct;16(10):2072-6.

Blood and Buccal DNA-Comparison: Abraham JE, Maranian MJ, Spiteri I, Russell R, Ingle S, Luccarini C, Earl HM, Pharoah PP, Dunning AM, Caldas C. Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping. BMC Med Genomics. 2012 May 30;5:19.

Buccal DNA-Comparisons of methods: King IB, Satia-Abouta J, Thornquist MD, ..., Shattuck AL, Potter JD, White E. Buccal cell DNA yield, quality, and collection costs: comparison of methods for large-scale studies. Cancer Epidemiol Biomarkers Prev. 2002 Oct;11(10 Pt 1):1130-3.

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