Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.
The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.
Lee
J S
JS
Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden.
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D012150:Polymorphism, Restriction Fragment Length; D019914:Taq Polymerase
D004247:DNA; D011163:Hydroxymethylbilane Synthase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; C056435:GTGCAC-specific type II deoxyribonucleases