Single Strand Conformation Polymorphism (SSCP) Analysis

  • Kim Hung Leung
  • Shea Ping Yip
Part of the Springer Protocols Handbooks book series (SPH)

1. Introduction

Variations in DNA sequences underlie the differences among different members of the same species and also between different species. DNA sequence variations are usually known as polymorphisms if the commonest allele is less than 0.99 in a given population (1). DNA polymorphisms are widespread in many different species, particularly in humans (2,3). Examples include single nucle-otide polymorphisms (SNPs), microsatellites, minisatellites, small insertions/ deletions, and large insertions/deletions. DNA polymorphisms may not have any phenotypic effect at the protein level or at the level of the whole organism. On the other hand, they are usually called disease-causing or pathogenic mutations if they cause a change in the phenotype and results in a disease status. The frequencies of individual mutations are usually not high because of selection pressure against such less favorable base changes. It is thus important to study DNA sequence variations in various branches of...


Polymerase Chain Reaction Product Sequence Variation Single Strand Single Strand Conformation Polymorphism Polymerase Chain Reaction Fragment 
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.


  1. 1.
    Harris H (1980)Human Biochemical Genetics, 3rd revised ed., Elsevier/North-Holland Biomedical Press, Amsterdam, The NetherlandsGoogle Scholar
  2. 2.
    Sachidanandam R, Weissman D, Schmidt SC et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409:928–933PubMedCrossRefGoogle Scholar
  3. 3.
    Redon R, Ishikawa S, Fitch KR et al (2006) Global variation in copy number in the human genome. Nature 444:444–454PubMedCrossRefGoogle Scholar
  4. 4.
    Cotton RGH (1997) Mutation detection. Oxford University Press, OxfordGoogle Scholar
  5. 5.
    Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770PubMedCrossRefGoogle Scholar
  6. 6.
    Yip SP (2000) Single-tube multiplex PCR-SSCP analysis distinguishes seven common ABO alleles and readily identifies new alleles. Blood 95:1487–1492PubMedGoogle Scholar
  7. 7.
    Emanuel JR, Damico C, Ahn S, Bautista D, Costa J (1996) Highly sensitive non-radioactive single-strand conformational polymorphism. Detection of Ki-ras mutations. Diagn Mol Pathol 5:260–264PubMedCrossRefGoogle Scholar
  8. 8.
    Yip SP, Hopkinson DA, Whitehouse DB (1999) Improvement of SSCP analysis by use of denaturants. BioTechniques 27:20–24PubMedGoogle Scholar
  9. 9.
    Hayashi K, Yandell DW (1993) How sensitive is PCR-SSCP? Hum Mutat 2: 338–346PubMedCrossRefGoogle Scholar
  10. 10.
    Yip SP, Lovegrove JU, Rana NA, Hopkinson DA, Whitehouse DB (1999) Mapping recombination hotspots in human phosphoglucomutase (PGM1). Hum Mol Genet 8:1699–1706PubMedCrossRefGoogle Scholar
  11. 11.
    Yip SP, Fung LF, Lo ST (2004) Rapid detection of common southeast Asian beta-thalassemia mutations by nonisotopic multiplex PCR-SSCP analysis. Genet Test 8: 104–108PubMedGoogle Scholar
  12. 12.
    Lim KP (2005) Mutational analysis of RHO, RDS and PRPF31 genes in Chinese patients with retinitis pigmentosa. MPhil thesis, The Hong Kong Polytechnic University, Hong Kong SAR, ChinaGoogle Scholar
  13. 13.
    Liu Q, Sommer SS (1995) Restriction endonuclease fingerprinting (REF): a sensitive method for screening mutations in long, contiguous segments of DNA. Biotechniques 18:470–477PubMedGoogle Scholar
  14. 14.
    Kringen P, Egedal S, Pedersen JC, Harbitz TB, Tveit KM, Berg K, Borresen-Dale AL, Andersen TI (2002) BRCA1 mutation screening using restriction endonu-clease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system. Electrophoresis 23:4085–4091PubMedCrossRefGoogle Scholar
  15. 15.
    Kukita Y, Tahira T, Sommer SS, Hayashi K (1997) SSCP analysis of long DNA fragments in low pH gel. Hum Mutat 10:400–407PubMedCrossRefGoogle Scholar
  16. 16.
    Vidal-Puig A, Moller DE (1994) Comparative sensitivity of alternative single-strand conformation polymorphism (SSCP) methods. Biotechniques 17:490–496PubMedGoogle Scholar
  17. 17.
    Paccoud B, Bourguignon J, Diarra-Mehrpour M, Martin J P, Sesboüé R (1998) Transverse formamide gradients as a simple and easy way to optimize DNA sin-glestrand conformation polymorphism analysis. Nucleic Acids Res 26:2245–2246PubMedCrossRefGoogle Scholar
  18. 18.
    Blancé H, Valette C, Bellanné-Chantelot C (1997) Optimization of non isotopic PCR-single-strand conformation polymorphism analysis. Clin Chem 43:2190–2192Google Scholar
  19. 19.
    Mohabeer AJ, Hiti AL, Martin WJ (1991) Non-radioactive single strand conformation polymorphism (SSCP) using the Pharmacia “Phast system.” Nucleic Acids Res 19:3154PubMedCrossRefGoogle Scholar
  20. 20.
    Campos B, Diez O, Cortes J, Domenech M, Pericay C, Alonso C, Baiget M (2001) Conditions for single-strand conformation polymorphism (SSCP) analysis of BRCA1 gene using an automated electrophoresis unit. Clin Chem Lab Med 39: 401–404PubMedCrossRefGoogle Scholar
  21. 21.
    Makino R, Yazyu H, Kishimoto Y, Sekiya T, Hayashi K (1992) F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. PCR Methods Appl 2:10–13PubMedGoogle Scholar
  22. 22.
    Iwahana H, Yoshimoto K, Mizusawa N, Kudo E, Itakura M (1994) Multiple fluorescence-based PCR-SSCP analysis. BioTechniques 16:296–305PubMedGoogle Scholar
  23. 23.
    Inazuka M, Tahira T, Hayashi K (1996) One-tube post-PCR fluorescent labeling of DNA fragments. Genome Res 6:551–557PubMedCrossRefGoogle Scholar
  24. 24.
    Iwahana H, Adzuma K, Takahashi Y, Katashima R, Yoshimoto K, Itakura M (1995) Multiple fluorescence-based PCR-SSCP analysis with postlabeling. PCR Methods Appl 4:275–282PubMedGoogle Scholar
  25. 25.
    Inazuka M, Wenz HM, Sakabe M, Tahira T, Hayashi K (1997) A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis. Genome Res 7:1094–1103PubMedGoogle Scholar
  26. 26.
    Iwahana H, Fujimura M, Takahashi Y, Iwabuchi T, Yoshimoto K, Itakura M (1996) Multiple fluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products. Biotechniques 21:510–519PubMedGoogle Scholar
  27. 27.
    Baba S, Kukita Y, Higasa K, Tahira T, Hayashi K (2003) Single-stranded confor-mational polymorphism analysis using automated capillary array electrophoresis apparatuses. Biotechniques 34:746–750PubMedGoogle Scholar
  28. 28.
    Doi K, Doi H, Noiri E, Nakao A, Fujita T, Tokunaga K (2004) High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis. Electrophoresis 25:833–838PubMedCrossRefGoogle Scholar
  29. 29.
    Tian H, Jaquins-Gerstl A, Munro N, Trucco M, Brody LC, Landers JP (2000) Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: a fast, simple method for detection of common mutations in BRCA1 and BRCA2. Genomics 63:25–34PubMedCrossRefGoogle Scholar
  30. 30.
    Leren TP, Solberg K, Rodningen OK, Ose L, Tonstad S, Berg K (1993) Evaluation of running conditions for SSCP analysis: application of SSCP for detection of point mutations in the LDL receptor gene. PCR Methods Appl 3:159–162PubMedGoogle Scholar
  31. 31.
    Larsen LA, Christiansen M, Vuust J, Andersen PS (1999) High-throughput single-strand conformation polymorphism analysis by automated capillary electrophoresis: robust multiplex analysis and pattern-based identification of allelic variants. Hum Mutat 13:318–327PubMedCrossRefGoogle Scholar
  32. 32.
    Liu Q, Feng J, Buzin C, Wen C, Nozari G, Mengos A, Nguyen V, Liu J, Crawford L, Fujimura FK, Sommer SS (1999) Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity. Biotechniques 26:932–942PubMedGoogle Scholar
  33. 33.
    Sarkar G, Yoon HS, Sommer SS (1992) Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations. Genomics 13:441–443PubMedCrossRefGoogle Scholar
  34. 34.
    Liu Q, Feng J, Sommer SS (1996) Bi-directional dideoxy fingerprinting (Bi-ddF): a rapid method for quantitative detection of mutations in genomic regions of 300–600 bp. Hum Mol Genet 5:107–114PubMedCrossRefGoogle Scholar
  35. 35.
    Ellison J, Squires G, Crutchfield C, Goldman D (1994) Detection of mutations and polymorphisms using fluorescence-based dideoxy fingerprinting (F-ddF). Biotechniques 17:742–753PubMedGoogle Scholar
  36. 36.
    Shevchenko YO, Bale SJ, Compton JG (2000) Mutation screening using automated bidirectional dideoxy fingerprinting. Biotechniques 28:134–138PubMedGoogle Scholar
  37. 37.
    Keen J, Lester D, Inglehearn C, Curtis A, Bhattacharya S (1991) Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels. Trends Genet 7:5PubMedCrossRefGoogle Scholar
  38. 38.
    Ravnik-Glavac M, Glavac D, Dean M (1994) Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum Mol Genet 3:801–807PubMedCrossRefGoogle Scholar
  39. 39.
    Axton RA, Hanson IM, Love J, Seawright A, Prosser J, van Heyningen V (1997) Combined SSCP/heteroduplex analysis in the screening for PAX6 mutations. Mol Cell Probes 11:287–292PubMedCrossRefGoogle Scholar
  40. 40.
    Kozlowski P, Krzyzosiak WJ (2001) Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection. Nucleic Acids Res 29:e71PubMedCrossRefGoogle Scholar
  41. 41.
    Simonsen K, Dissing J, Rudbeck L, Schwartz M (1999) Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR-SSCP: detection of a new polymorphic mutation. Ann Hum Genet 63:193–197PubMedCrossRefGoogle Scholar
  42. 42.
    Tsai MY, Suess P, Schwichtenberg K, Eckfeldt JH, Yuan J, Tuchman M, Hunninghake D (1993) Determination of apolipoprotein E genotypes by single-strand conformational polymorphism. Clin Chem 39:2121–2124PubMedGoogle Scholar
  43. 43.
    Daly AK, King BP, Leathart JB (2006) Genotyping for cytochrome P450 polymorphisms. Methods Mol Biol 320:193–207PubMedGoogle Scholar
  44. 44.
    Wilton SD, Honeyman K, Fletcher S, Laing NG (1998) Snapback SSCP analysis: engineered conformation changes for the rapid typing of known mutations. Hum Mutat 11:252–258PubMedCrossRefGoogle Scholar
  45. 45.
    Quaranta S, Chevalier D, Bourgarel-Rey V et al (2006) Identification by single-strand conformational polymorphism analysis of known and new mutations of the CYP3A5 gene in a French population. Toxicol Lett 164:177–184PubMedCrossRefGoogle Scholar
  46. 46.
    Kamio K, Matsushita I, Tanaka G, Ohashi J, Hijikata M, Nakata K, Tokunaga K, Azuma A, Kudoh S, Keicho N (2004) Direct determination of MUC5B promoter haplotypes based on the method of single-strand conformation polymorphism and their statistical estimation. Genomics 84:613–622PubMedCrossRefGoogle Scholar
  47. 47.
    Beheshti I, Hanson NQ, Copeland KR, Garg U, Tsai MY (1995) Single-strand conformational polymorphisms (SSCP): studies of the genetic polymorphisms of exon 4 of apolipoprotein C III. Clin Biochem 28:303–307PubMedCrossRefGoogle Scholar
  48. 48.
    Clarridge JE III (2004) Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17:840–862PubMedCrossRefGoogle Scholar
  49. 49.
    Widjojoatmodjo MN, Fluit AC, Verhoef J (1994) Rapid identification of bacteria by PCR-single-strand conformation polymorphism. J Clin Microbiol 32:3002–3007PubMedGoogle Scholar
  50. 50.
    Turenne CY, Witwicki E, Hoban DJ, Karlowsky JA, Kabani AM (2000) Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 38:513–520PubMedGoogle Scholar
  51. 51.
    Gillman LM, Gunton J, Turenne CY, Wolfe J, Kabani AM (2001) Identification of Mycobacterium species by multiple-fluorescence PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 39:3085–3091PubMedCrossRefGoogle Scholar
  52. 52.
    Schwieger F, Tebbe CC (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64:4870–4876PubMedGoogle Scholar
  53. 53.
    King S, McCord BR, Riefler RG (2005) Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soil bacteria. J Microbiol Methods 60:83–92PubMedCrossRefGoogle Scholar
  54. 54.
    Walsh TJ, Francesconi A, Kasai M, Chanock SJ (1995) PCR and single-strand conformational polymorphism for recognition of medically important opportunistic fungi. J Clin Microbiol 33:3216–3220PubMedGoogle Scholar
  55. 55.
    Kumar M, Shukla PK (2006) Single-stranded conformation polymorphism of large subunit of ribosomal RNA is best suited to diagnosing fungal infections and differentiating fungi at species level. Diagn Microbiol Infect Dis 56:45–51PubMedCrossRefGoogle Scholar
  56. 56.
    Soares CC, Volotao EM, Albuquerque MC, Nozawa CM, Linhares RE, Volokhov D, Chizhikov V, Lu X, Erdman D, Santos N (2004) Genotyping of enteric adeno-viruses by using single-stranded conformation polymorphism analysis and heter-oduplex mobility assay. J Clin Microbiol 42:1723–1726PubMedCrossRefGoogle Scholar
  57. 57.
    Golijow CD, Perez LO, Smith JS, Abba MC (2005) Human papillomavirus DNA detection and typing in male urine samples from a high-risk population from Argentina. J Virol Methods 124:217–220PubMedCrossRefGoogle Scholar
  58. 58.
    Mackiewicz V, Roque-Afonso AM, Marchadier E, Nicand E, Fki-Berrajah L, Dussaix E (2005) Rapid investigation of hepatitis A virus outbreak by single strand conformation polymorphism analysis. J Med Virol 76:271–278PubMedCrossRefGoogle Scholar
  59. 59.
    Telenti A, Honore N, Cole ST (1998) Detection of mutations in mycobacteria by PCR-SSCP (single-strand conformation polymorphism). Methods Mol Biol 101: 423–430PubMedGoogle Scholar
  60. 60.
    Kim BJ, Lee KH, Yun YJ, Park EM, Park YG, Bai GH, Cha CY, Kook YH (2004) Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene (rpoB). J Microbiol Methods 58:111–118PubMedCrossRefGoogle Scholar
  61. 61.
    Beckmann L, Muller M, Luber P, Schrader C, Bartelt E, Klein G (2004) Analysis of gyrA mutations in quinolone-resistant and -susceptible Campylobacter jejuni isolates from retail poultry and human clinical isolates by non-radioactive single-strand conformation polymorphism analysis and DNA sequencing. J Appl Microbiol 96:1040–1047PubMedCrossRefGoogle Scholar
  62. 62.
    Cordell HJ, Clayton DG (2005) Genetic association studies. Lancet 366:1121–1131PubMedCrossRefGoogle Scholar
  63. 63.
    Norton N, Williams NM, O'Donovan MC, Owen MJ (2004) DNA pooling as a tool for large-scale association studies in complex traits. Ann Med 36:146–152PubMedCrossRefGoogle Scholar
  64. 64.
    Sasaki T, Tahira T, Suzuki A, Higasa K, Kukita Y, Baba S, Hayashi K (2001) Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA. Am J Hum Genet 68:214–218PubMedCrossRefGoogle Scholar
  65. 65.
    Higasa K, Kukita Y, Baba S, Hayashi K (2202) Software for machine-independent quantitative interpretation of SSCP in capillary array electrophoresis (QUISCA). Biotechniques 33:1342–1348Google Scholar
  66. 66.
    Tahira T, Okazaki Y, Miura K, Yoshinaga A, Masumoto K, Higasa K, Kukita Y, Hayashi K (2006) QSNPlite, a software system for quantitative analysis of SNPs based on capillary array SSCP analysis. Electrophoresis 27:3869–3878PubMedCrossRefGoogle Scholar
  67. 67.
    Gasser RB, Chilton NB (2001) Applications of single-strand conformation polymorphism (SSCP) to taxonomy, diagnosis, population genetics and molecular evolution of parasitic nematodes. Vet Parasitol 101:201–213PubMedCrossRefGoogle Scholar
  68. 68.
    Bosio CF, Harrington LC, Jones JW, Sithiprasasna R, Norris DE, Scott TW (2005) Genetic structure of Aedes aegypti populations in Thailand using mitochondrial DNA. Am J Trop Med Hyg 72:434–442PubMedGoogle Scholar
  69. 69.
    Godinho R, Domingues V, Crespo EG, Ferrand N (2006) Extensive intraspecific polymorphism detected by SSCP at the nuclear C-mos gene in the endemic Iberian lizard Lacerta schreiberi. Mol Ecol 15:731–738PubMedCrossRefGoogle Scholar
  70. 70.
    Wan QH, Zhu L, Wu H, Fang SG (2006) Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca). Mol Ecol 15:2441–2450PubMedCrossRefGoogle Scholar
  71. 71.
    Di Gaspero G, Cipriani G (2003) Nucleotide binding site/leucine-rich repeats, Pto-like and receptor-like kinases related to disease resistance in grapevine. Mol Genet Genomics 269:612–623PubMedCrossRefGoogle Scholar
  72. 72.
    Baldi P, Patocchi A, Zini E, Toller C, Velasco R, Komjanc M (2004) Cloning and linkage mapping of resistance gene homologues in apple. Theor Appl Genet 109: 231–239PubMedCrossRefGoogle Scholar
  73. 73.
    Ohsako T, Wang GZ, Miyashita NT (1996) Polymerase chain reaction-single strand conformational polymorphism analysis of intra- and interspecific variations in organellar DNA regions of Aegilops mutica and related species. Genes Genet Syst 71:281–292PubMedCrossRefGoogle Scholar
  74. 74.
    Sunnucks P, Wilson AC, Beheregaray LB, Zenger K, French J, Taylor AC (2000) SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol Ecol 9: 1699–1710PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Kim Hung Leung
    • 1
  • Shea Ping Yip
    • 1
  1. 1.Department of Health Technology and InformaticsThe Hong Kong Polytechnic UniversityKowloonChina

Personalised recommendations