title: Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.
abstract: In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, 47907.
authors.forename: Kushani
authors.initials: K
authors.lastname: Shah
authors.forename: Shelby
authors.initials: S
authors.lastname: Thomas
authors.forename: Arnold
authors.initials: A
authors.lastname: Stein
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000193:education*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012106:Research / Q000193:education; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D013334:Students; D013663:Teaching / Q000379:methods*; D014495:Universities
chemical_list: D004247:DNA
keywords: DNA sequencing; genotyping; single nucleotide polymorphisms