Translation of "Template strand" in German

The resulting 3'- and/or 2'-amino- and thiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of complementary strands in the presence of a template strand or of oligonucleotides and for the detection of genetic material.

SUMMARY OF THE INVENTION It has now been found that a fluorescent dye can be coupled via an amino or thiol group in the 3'-(2') position (in a or ? position) of a nucleoside, nucleotide or oligonucleotide, and this compound can advantageously be used to synthesize complementary strands in the presence of a template strand or of oligonucleotides, and to detect genetic material in vivo and in vitro.

It has now been found that a fluorescent dye can be coupled via an amino or thiol group in the 3'-(2') position (in a or ? position) of a nucleoside, nucleotide or oligonucleotide, and this compound can advantageously be used to synthesize complementary strands in the presence of a template strand or of oligonucleotides, and to detect genetic material in vivo and in vitro.

Starting from a compound of the formula I which has a triphosphate in the 5' position, it is possible to carry out the synthesis of the DNA double strand using any suitable primer and a template strand with the aid of a polymerase, i.e. an enzyme which in the presence of suitable substrates synthesizes a true complementary copy of the sequence, in the presence of the four nucleoside triphosphates, preferably with the aid of T7 or Taq polymerase, DNA polymerase I and reverse transcriptase.

A disadvantage of the use of the Klenow polymerase for amplifying nucleic acids using PNA/DNA primers is that this enzyme is not sufficiently temperature-stable that, for example, an amplification of the template strand copied can be achieved analogously to known amplification techniques such as PCR and LCR.

For amplification of the template strand with the aid of the VENT™ DNA polymerase and of the PNA/DNA primer 1, an excess of primer of 20 pmol of PNA/DNA primer 1 was mixed with 2 pmol of DNA template 3 (81 -mer) and 200 mM dNTPs in a final volume of 50 ?l of suitable reaction buffer.

Then, if necessary, the newly synthesized strand of nucleic acid can be separated from the template strand for processing in Step (d).

PCR exploits the properties of the enzyme DNA polymerase to synthesize a complementary strand of DNA from a template strand.

This hairpin structure is thought to stabilize the misaligned DNA strands and after a further round of replication, the result is an increase in the number of repeat sequences (a repeat expansion) if the hairpin formed on the 'lagging' strand of DNA, or a decrease in the number of repeat sequences (a repeat contraction) if the hairpin formed on the 'template' strand of DNA.

Processing optionally carried out in the following Step (d), is preferably a separation of the newly synthesized strand of DNA from the template strand.

Treatment that may perhaps be carried out in a subsequent Step (d) is preferably the separation of the newly synthesized DNA strand from the template strand.

Polymerase II appears to facilitate DNA synthesis which starts from a damaged template strand and thus preserves mutations.

The processing initially includes a separation of the newly synthesized strand of nucleic acid from the template strand, whereby the strand separation is preferably carried out thermally, enzymatically and/or chemically.

The above-mentioned primers i.e. short-chain oligonucleotides should form stable base pairs with the template strand as well as not be attacked by endogenous nucleases in order to ensure an optimal function.

This process has significant effects on the metabolism of lipoproteins, and it necessitates a single-strand template with clear-cut characteristics in the vicinity of the edited base.

The termination of the synthesis can be specifically determined in each case by using a 3'-(2')-amino-modified A,C,G,T nucleotide of the formula I. This is particularly important for the synthesis of DNA complementary strands in the presence of a template strand and thus also for the sequencing of DNA strands, because the use of a modified nucleotide ensures very base-specific termination of the reaction.

A treatment potentially carried out in a subsequent Step (d) is preferably a separation of the newly synthesized DNA strands from the template strand.

Denaturing of the newly synthesized strand of nucleic acid and template strand follows, and second strand synthesis is then carried out when a double-strand sequence recognizable with a restriction endonuclease is formed.

Denaturing of the newly synthesized strand of nucleic acid and template strand is subsequently carried out by second-strand synthesis, which results in a double strand sequence recognizable by a restriction endonuclease.