Two different methods of an amplification of DNA:
Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes.
To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a plasmid.
A plasmid (also called DNA cloning vector in this context) is a small circular DNA molecule that replicates independently of the chromosomal DNA in bacteria. The plasmid molecules can be used to provide a "vehicle" in which to insert a desired DNA fragment.
Modified plasmids are usually reintroduced into a bacterial host for replication.
As the bacteria divide, they copy their own DNA (including the plasmids).
The plasmid cloning vectors contain many short DNA sequences that can be cut with different commonly available restriction enzymes.
Restriction enzymes (also called restriction endonucleases) recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA.
Many restriction enzymes make staggered cuts in the two strands of DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang.
The sequence that is recognized by the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome.
The unpaired nucleotides remaining on a single strand at each end of the restriction fragment, often referred to as sticky ends, can then form base pairs with other short strands having a complementary sequence.
The process of forming hydrogen bonds between complementary sequences on single strands to form double- stranded DNA is called annealing.
DNA ligase can then seal the gap in each strand in the new DNA molecule.
In this way, researchers can produce recombinant DNA by joining DNA from two different sources.
Polymerase chain reaction (PCR) is a technique used to rapidly increase the number of copies of specific regions of DNA for further analyses.
Polymerase chain reaction process quickly rises the number of DNA fragments
PCR uses a special form of DNA polymerase, the enzyme that replicates DNA, and other short nucleotide sequences called primers that base pair to a specific portion of the DNA being replicated.
The primer nucleotides have a sequence that is complementary to the 3′ end of each strand of the sample DNA molecule on either side of the DNA target sequence.
Polymerase chain reaction steps
Polymerase chain reaction is used for many purposes in laboratories.
Applications of polymerase chain reaction include:
- the identification of the owner of a DNA sample left at a crime scene;
- paternity analysis;
- the comparison of small amounts of ancient DNA with modern organisms;
- determining the sequence of nucleotides in a specific region.
Articles related to “Methods in molecular genetics”:
- DNA Amplification: DNA cloning and polymerase chain reaction (PCR)
- DNA Isolation (extraction steps) and Sorting (agarose gel electrophoresis)
- Genetic engineering - Recombinant DNA technology steps
- Types of DNA sequencing: Sanger sequencing, whole genome shotgun sequencing, next-generation sequencing