Improved cycle sequencing of GC-rich templates by a combination of nucleotide analogs.

A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.

Direct DNA sequence determination from total genomic DNA.

It is possible to perform a combined amplification and sequencing reaction ('DEXAS') directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily determined directly from total genomic DNA.

Direct exponential amplification and sequencing (DEXAS) of genomic DNA.

In order to supply a sufficient amount of template molecules for DNA sequence determination, cloning into plasmids and subsequent plasmid purification, or amplification via the PCR, are generally used. Here, we present a method-'direct exponential amplification and sequencing' or 'DEXAS'-that permits direct sequence determination from whole genomic DNA and thus eliminates the need for template amplification and preparation. It relies on the simultaneous amplification of a target sequence and the determination of its sequence using dideoxyterminators in a two-step cycling reaction. DEXAS can be applied to single as well as multi-copy genomic sequences, and can easily be automated.

Screening for mitochondrial DNA (mtDNA) point mutations using nonradioactive single strand conformation polymorphism (SSCP) analysis.

OBJECTIVES: Mitochondrial cytopathies such as Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), and myoclonus epilepsy with red ragged fibers (MERRF) are associated with distinct mtDNA point mutations (for review see 1). LHON, for example, is related to at least 14 mtDNA point mutations within different mitochondrially encoded respiratory subunit genes. In addition, the number of newly found LHON-related mutations is increasing. In the light of the large number and the dispersed distribution of these point mutations throughout the mitochondrial genome, screening for these by sequencing all of suspected loci is laborious and time-consuming. In order to facilitate a rapid screening for mitochondrial point mutations we have evaluated the use of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for the analysis of the human mitochondrial genome. DESIGN AND METHODS: In a first evaluation step we created a variety of pUC18 clones derived from mitochondrial control region amplifications with defined sequence differences and length. These clones were used as standard material for an optimization of the PCR-SSCP analysis. The optimized PCR-SSCP was then applied to large cohorts of patients with known, i.e., sequenced mtDNA point mutations and to healthy controls in order to evaluate its sensitivity. RESULTS: The most common LHON-related mtDNA point mutations at nucleotide positions (nps) 11778, 14484, 4216, could be detected by SSCP analysis, as well as the heteroplasmic np 3243 MELAS associated point mutation. Several new polymorphisms and point mutations were found. A sensitivity, i.e., the ability to detect defined point mutations, of 93% (clones) and 98% (disease controls) was achieved when comparing SSCP- and direct sequencing results. CONCLUSION: The PCR-SSCP approach using a non-radioactive silver staining method is suited for the detection of human mitochondrial point mutations, as well as a helpful screening tool for novel mt DNA mutations.

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.