<P> On the other hand, many of the in vitro laboratory techniques that involve DNA polymerase in biochemistry and molecular biology (such as DNA sequencing and the polymerase chain reaction), use DNA primers because they are more temperature stable . In experiments, it is often important to use a primer with a similar Tm (melting temperature) to the template strand it will be hybridizing to . A primer with a Tm significantly higher than the reaction's annealing temperature may mishybridize and extend at an incorrect location along the DNA sequence, while one with a Tm significantly lower than the annealing temperature may fail to anneal and extend at all . These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases . They are hybridized to a target DNA, which is then copied by the polymerase . </P> <P> The lagging strand of DNA is that strand of the DNA double helix that is orientated in a 5 ′ to 3 ′ manner . Therefore, its complement must be synthesized in a 3 ′ → 5 ′ manner . Because DNA polymerase III cannot synthesize in the 5 ′ → 3 ′ direction, the lagging strand is synthesized in short segments known as Okazaki fragments . Along the lagging strand's template, primase builds RNA primers in short bursts . DNA polymerases are then able to use the free 3 ′ - OH groups on the RNA primers to synthesize DNA in the 5 ′ → 3 ′ direction . </P> <P> The RNA fragments are then removed by DNA polymerase I for prokaryotes or DNA polymerase δ for eukaryotes (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present . DNA ligase then joins the deoxyribonucleotides together, completing the synthesis of the lagging strand . </P> <P> In eukaryotic primer removal, DNA polymerase δ extends the Okazaki fragment in 5 ′ to 3 ′ direction, and when it encounters the RNA primer from the previous Okazaki fragment, it displaces the 5 ′ end of the primer into a single - stranded RNA flap, which is removed by nuclease cleavage . Cleavage of the RNA flaps involves either flap structure - specific endonuclease 1 (FEN1) cleavage of short flaps, or coating of long flaps by the single - stranded DNA binding protein replication protein A (RPA) and sequential cleavage by Dna2 nuclease and FEN1 . </P>

What is the purpose of the primer in dna sequencing