John M. S. Bartlett.pdf - Bio-Nica.info

Digoxigenin 347
51
Nonradioactive PCR Sequencing Using Digoxigenin
Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger,
and Manuel B. Graeber
1. Introduction
Techniques for direct sequencing of polymerase chain reaction (PCR) products are
of central importance to contemporary research in molecular biology and genetics. The
rapidly growing number of cloned human disease genes increasingly allows sequencing
of PCR amplicons for diagnostic purposes. Nonradioactive sequencing protocols are of
particular use because health, environmental, and administrative risks are minimized
compared with conventional isotopic methods. The PCR-based nonradioactive cycle
sequencing protocol described in this chapter has been successfully used to sequence
mitochondrial and nuclear genes in Parkinson’s and Alzheimer’s disease brains using
DNA extracted from formalin-fixed and paraffin-embedded neuropathological material
(1–3). This method, which allows sequence information of PCR products to be
obtained within a single day, can be performed in a research or clinical laboratory using
relatively inexpensive equipment.After initial PCR amplification, amplicons are purified
using spin columns for affinity chromatography or ultrafiltration. Subsequently, cycle
sequencing (4) is performed using 5′-digoxigenin end-labeled oligonucleotide primers.
Because the nucleotide sequences of the PCR and sequencing primers can be identical,
both reactions may be performed using the same thermalcycling protocol. This obviates
the need for time-consuming optimization procedures. For visualization of sequencing
results, sequencing reactions are separated on a standard sequencing gel, the gel is
contact-blotted to a nylon membrane, and sequencing bands are visualized using
alkaline phosphatase-conjugated antibodies (Fig. 1).
Potential pitfalls of our method are primarily related to the extreme sensitivity of PCR.
The need for positive and negative sample controls cannot be overemphasized. We use
different rooms and different pipets for setting up PCRs, pipetting sequencing templates,
and thermal cycling (5). In addition, aerosol-resistant pipet tips are always used.
2. Materials
2.1. Purification of Sequencing Templates
1. 10× TNE: 100 mM Tris-HCl, pH 7.4, 1.0 M NaCl, 10 mM EDTA (see Note 1).
2. Wizard PCR Preps DNA Purification System containing affinity chromatography spin
columns, purification buffer, and resin (A7170, Promega, Madison, WI). Not contained
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
347