Question

introduction about Basic techniques and DNA synthesis ( maximum 2 pages )

Answer 1

DNA synthesis definition

DNA synthesis is defined as the process by which copies of nucleic acids are strung together to form a longer DNA sequence within a laboratory setting.

• DNA replication is semiconservative. Each strand in the double helix acts as a template for synthesis of a new, complementary strand.

• New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5' to 3' direction.

• During DNA replication, one new strand (the leading strand) is made as a continuous piece. The other (the lagging strand) is made in small pieces.

• DNA replication requires other enzymes in addition to DNA polymerase, including DNA primase, DNA helicase, DNA ligase, and topoisomerase.

DNA polymerase

One of the key molecules in DNA replication is the enzyme DNA polymerase. DNA polymerases are responsible for synthesizing DNA: they add nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template.

Starting DNA replication

Replication always starts at specific locations on the DNA, which are called origins of replication and are recognized by their sequence.

E. coli, like most bacteria, has a single origin of replication on its chromosome. The origin is about 245245245245 base pairs long and has mostly A/T base pairs (which are held together by fewer hydrogen bonds than G/C base pairs), making the DNA strands easier to separate.

Specialized proteins recognize the origin, bind to this site, and open up the DNA. As the DNA opens, two Y-shaped structures called replication forks are formed, together making up what's called a replication bubble. The replication forks will move in opposite directions as replication proceeds.

Primers and primase

DNA polymerases can only add nucleotides to the 3' end of an existing DNA strand. (They use the free -OH group found at the 3' end as a "hook," adding a nucleotide to this group in the polymerization reaction.)

Primase makes an RNA primer, or short stretch of nucleic acid complementary to the template, that provides a 3' end for DNA polymerase to work on. A typical primer is about five to ten nucleotides long. The primer primes DNA synthesis, i.e., gets it started. Once the RNA primer is in place, DNA polymerase "extends" it, adding nucleotides one by one to make a new DNA strand that's complementary to the template strand.

Leading and lagging strands

In E. coli, the DNA polymerase that handles most of the synthesis is DNA polymerase III. There are two molecules of DNA polymerase III at a replication fork, each of them hard at work on one of the two new DNA strands.

DNA polymerases can only make DNA in the 5' to 3' direction, and this poses a problem during replication. A DNA double helix is always anti-parallel; in other words, one strand runs in the 5' to 3' direction, while the other runs in the 3' to 5' direction. This makes it necessary for the two new strands, which are also antiparallel to their templates, to be made in slightly different ways.