Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
Polymerase chain reaction is method of obtaining large quantities of DNA sequence by selective replication. The basic components of PCR-setup are- DNA polymerase, DNA template, two DNA primers, nucleotides and buffer. The three temperature steps for one cycle are - denaturation, primer annealing and extension steps.
There are two methods for visualizing the PCR products-
1.Staining of amplified DNA product with chemical dye such as ethidium bromide.
2.Labeling the PCR primers or nucleotides with fluorescent dye prior to amplification.
The mostly widely use method to analyze PCR product is agarose gel electrophoresis which separate the DNA fragments based on size and charge. As DNA is negatively charged, it will move towards positively charged electrode. As the gel runs, the shorter DNA fragment will move faster than the longer ones because it has less resistance when moving through gel pores. The DNA fragments can be seen as the bands, each representing a group of same sized DNA fragments. A single band contain 1000s of DNA bases of same length.
In PCR screening, the first lane of a gel is loaded with the molecular weight marker(DNA ladder). Then there are lanes with DNA of samples containing gene of interest. There is positive control lane, with known DNA sequence. It is to match our samples to the control DNA.The other lanes , where no bands are seen , do not contain the known DNA sequence.
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