Structural Biochemistry/DNA recombinant techniques

Overview

DNA recombinant techniques merely refer to processes that scientists use to manipulate, duplicate and apply DNA for research. Deletions, insertions and substitutions are the most useful changes implemented for the synthesis of new genes. Such techniques usually include the cloning or amplification of sequences for further study. Restriction enzymes and DNA ligase play a vital role in the in the production of recombinant DNA. Polymerase chain reaction (PCR) is often used on interested sequences to be cut by nucleases and then cloned by phages, BACs or YACs. Using a DNA or RNA probe, specific genes can be cloned from a genomic library. The usage of various recombinant techniques can lead scientists to new understandings of how DNA affects us and other organisms.

Vectors

A vector is a DNA molecule that can be used to insert a DNA sequence into a cell. Vectors are used for replication. Usual vectors in laboratories are plasmids or viral DNA (Lambda Phage). Vectors must be capable of being replicated by the cell.

Plasmid Vectors

Plasmids are naturally occurring circular pieces of DNA that are found in bacteria. Plasmids can be used as vectors to incorporate and replicate a DNA insert of interest by joining that DNA insert into the plasmid DNA. The vector is prepared to accept a DNA insert by treatment with a restriction enzyme (like EcoRI), which cleaves it at specific sites and leaves complementary single stranded "sticky ends." The DNA fragment to be inserted is treated with the same restriction enzyme so that it has complementary ends to those of the vector. DNA ligase is then used to join the DNA insert into the vector plasmid, resulting in recombination. The recombinant plasmid DNA can be cloned and amplified as the bacteria host colony grows.

Enzymes used in Recombinant DNA Technology

1. Type II restriction endonucleases always recognize palindromic sequences and the are able to cleave the DNA strand. In other words, they break the phosphodiester bonds of specific base sequences in the DNA.
2. DNA ligase is the opposite of Type II restriction endonuclease in that it joins two DNA molecules or fragments together.
3. DNA Polymerase is an enzyme that is able to use a template DNA and synthesize a complimentary strand by adding nucleotides to the 3' ends.
4. Reverse transcriptase is an enzyme that creates a DNA from an RNA molecule. Reverse transcriptase does the opposite of RNA polymerase, which takes DNA and makes RNA.
5. Polynucleotide kinase adds a phosphate to the 5' -OH end of a polynucleotide. it is useful in radioactively label DNA or permit ligation.