DNases chain extension

Lamivudine (Epivir-HBV, 3TC) is a nucleoside analog that competitively inhibits viral reverse transcriptase and terminates pro viral DNA chain extension. Because it does not affect host response, it suppresses viral replication but does not directly eliminate the virus from the hep-atocy tes. Its efficacy is also not associated with, or dependent upon, the flare response seen with interferon. Like IFN-a2b though, lamivudine demonstrates higher response rates in patients with elevated transaminases (ALT >100) and lower viral loads. ... 

Also present in the first test tube is a synthetic analog of ATP in which both the 2 and 3 hydroxyl groups have been replaced by hydrogens This compound is called 2 3 dideoxyadenosme triphosphate (ddATP) Similarly ddTTP is added to the second tube ddGTP to the third and ddCTP to the fourth Each tube also contains a primer The primer is a short section of the complementary DNA strand which has been labeled with a radioactive isotope of phosphorus ( P) When the electrophoresis gel is examined at the end of the experiment the positions of the DNAs formed by chain extension of the primer are located by a technique called autoradiography which detects the particles emitted by the P isotope... 

Initially, the double-stranded DNA is heated to separate the strands. The primers are then added and the temperature lowered so that the primers anneal to the complementary sequences of each strand. In the presence of nucleoside triphosphates, the DNA polymerase enzyme will replicate a length of DNA starting from the 3 -end of a nucleotide, extending the chain towards the 5 -end (see Section 14.2.2). It will thus start chain extension from the 3 -ends of the primers and continue to the end of the DNA strands. This will lead to two double-stranded DNA... 

The process is repeated. Heating causes separation of strands, and cooling allows primer to attach to the appropriate nucleotide sequence. Enzymic chain extension then produces four double-stranded DNA molecules. 

This enzyme [EC 2.7.7.49], also known as RNA-directed DNA polymerase, DNA nucleotidyltransferase (RNA-directed), and revertase, catalyzes the RNA-template-directed extension of the 3 -end of a DNA strand by one deoxynucleotide at a time n deoxynucleoside triphosphate to produce n pyrophosphate (or, diphosphate) and DNA . The enzyme cannot initiate a DNA chain de novo and requires a DNA or RNA primer. See also Viral Polymerases... 

Since eukaryotic chromosomes are linear, the ends of these chromosomes require a special solution to ensure complete replication. This can be seen in figure 26.26. At the very end of a linear duplex a primer is necessary to initiate DNA replication. After RNA primer removal there is bound to be a gap at the 5 end of the newly synthesized DNA chains. Since DNA synthesis always requires a primer the usual way of filling this gap is not going to solve the problem. This dilemma is overcome by a special structure at the ends (telomeres) of eukaryotic chromosomes and a special type of reverse transcriptase (telomerase) that synthesizes telomeric DNA. In many eukaryotes the telomeres contain short sequences (frequently hexamers) that are tan-demly repeated many times. Telomerase contains an RNA that binds to the 3 ends and also serves as a template for the extension of these ends. Prior to replication, the 3 ends of the chromosome are extended with additional tandemly repeated hexamers. The 3 ends are extended sufficiently so that there is room to accommodate an RNA primer. In this way there is no net loss of DNA from the 5 ends as a result of replication. After replication the 3 end is somewhat... 

Fig. 1.1. Mechanism of chain extension catalysed by DNA polymerase on a primed template. (For discussion see text.)...

Fig. 2.1. Chain extension reaction catalysed by DNA polymerase I. Chain growth occurs in the 5 - 3 direction by the stepwise addition of nucleotides to the free 3 -OH end of the primer strand.

Fig. 2.5. Priming with a long Hae HI restriction fragment on single-stranded circular DNA. After the chain extension step digestion with Hae III removes the unlabelled primer from the heterogeneous set of labelled extension sequences.

The primers used are restriction fragments which originated from within the duplex DNA to be sequenced. In the annealing reaction, to produce the primer-template complex, only one of the primer strands is hybridized to its complementary sequence in the template since the other potential template is destroyed by the Exo III treatment. The result is the formation of a primer-template which can be extended by DNA polymerase in a direction opposite to that of the Exonuclease III attack. This is shown in Fig. 3.15. Two inherent features of the method which can cause problems are also made clear in Fig. 3.15. In the first place priming and chain extension can also occur on the 3 -ends of both template strands when sufficient complementarity remains between the 3 -ends of the template strands to form a base-paired structure. Secondly, a primer which originates from near the centre of the duplex DNA... 

After completion of the chain extension reaction (above) the addition of EDTA is omitted and 1 unit of the restriction enzyme added to each capillary which is then incubated for a further 5 min. at 37°C. The small amount of DNA in each sample should be... 

Alternative chain extension reaction using reverse transcriptase Reverse transcriptase can be used as an effective alternative to DNA polymerase (Klenow subfragment) in the chain-termination reaction. The following protocol is taken from Smith (1980). 

Plate III. A sequencing gel showing the patterns obtained from eight M13mp2 recombinant phage DNAs picked at random. Chain extension was carried out in the presence of ddT only to identify recombinants containing identical cloned... 

Two types of primer DNA (Sections 4.2.1. and 4.8.1.) may be employed for the chain-extension reaction. When the desired sequence, of chain length up to —400 nucleotides, is cloned into M13mp2, a flanking primer which hybridizes to a region of the... 

---