Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.
Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for beta-thalassemia.
Salk
Jesse J
JJ
Department of Biology, Brandeis University, Waltham, MA 02454, USA. jsalk@fhcrc.org
Sanchez
J Aquiles
JA
Pierce
Kenneth E
KE
Rice
John E
JE
Soares
Kevin C
KC
Wangh
Lawrence J
LJ
D002460:Cell Line; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000032:analysis*; D014644:Genetic Variation; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA / Q000379:methods
D004277:DNA, Single-Stranded