Direct DNA sequence determination from total genomic DNA
Direct DNA sequence determination from total genomic DNAChristian Kilger* and Svante Pääbo
Institute of Zoology, University of Munich, PO Box 202126, D-80021 Munich, Germany
Received February 11, 1997;Revised and Accepted April 1, 1997
ABSTRACT
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.
Cycle sequencing' (1 ) generates a sequence reaction during repeated cycles of thermal denaturation and extension of an oligonucleotide primer using a thermostable DNA polymerase. One of its advantages is that it requires drastically less template DNA than other DNA sequencing approaches. However, since it does not exponentially amplify the target sequence, as is achieved by the polymerase chain reaction (PCR) (2 ), it has hitherto been impossible to use cycle sequencing procedures to directly determine nucleotide sequences from complex genomes. Attempts to develop such methods have yielded two-stage protocols, e.g. `coupled amplification and sequencing' or CAS (3 ,4 ), where in a first stage a sequencing template is generated by PCR, and in a second stage sequencing as well as additional amplification of the target sequence is achieved. Recently, a further development, `direct exponential amplification and sequencing' or DEXAS (5 ), achieved the amplification of the template DNA as well as the sequencing reaction simultaneously in a single reaction, using two primers, a mixture of deoxynucleotides (dNTP) and dideoxynucleotides (ddNTP), and a thermostable DNA polymerase carrying the F667Y mutation (6 ) (e.g. ThermoSequenaseTM, TaquenaseTM, AmpliTaqFSTM), which allows the enzyme to incorporate ddNTPs with an efficiency approaching that of dNTPs. Of importance for the generation of readable sequences by DEXAS is that unequal ratios of the two primers are used and that these are of sufficient length (>25mers) to allow a high temperature to be maintained throughout the reaction. However, in spite of these measures, impractically high amounts of genomic DNA (>0.8 [mu]g) are needed to determine single-copy sequences in the human genome by DEXAS (5 ).
We speculated that the requirement for large amounts of template DNA in DEXAS might be due to the fact that many extension products are terminated through the incorporation of a ddNTP rather than extended to the other primer binding site, thereby stifling the exponential amplification of the target sequence. Therefore, we investigated whether a combination of two DNA polymerases that differ in their ability to incorporate ddNTPs might solve this problem by uncoupling the production of templates from the production of sequencing ladders (Fig. 1 ).
