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.
Protein Agarose Gel Electrophoresis
Objectives: At the end of the exercise, you should be able to
1. Understand the concepts of protein structures, isoelectric point of amino acids and
proteins.
2. Understand the principle of separation in protein agarose gel electrophoresis.
3. Demonstrate laboratory techniques used in running a protein agarose gel (preparing
gels, loading samples with micropippets, and electrophoresis).
4. Understand the molecular difference between normal and mutant forms of betahemoglobin in patients with sickle cell disease, and how protein agarose gel
electrophoresis can be used in disease diagnosis.
Comparing Migration Rates of Hemoglobin Isolated from Normal, Heterozygous
and Sickle cell anemia Individuals at pH 9.2: Part B: pH 9.2, 1X buffer
Materials on the supply bench:
1) gel running apparatus (lid matches the tank); one comb.
2) flasks for preparing agarose gel (125 ml)
3) micropippets (P20) and yellow tip boxes
4) plastic waste cup, markers and microcentrifuge tubes
5) pH 8.6 buffer for part A
6) pH 9.2 buffer for part B
7) cylinders
8) 4 protein samples for part A (on ice)
9) 3 protein samples for part B (on ice)
10) agarose powder, balance
Part B Samples to be loaded into the pH 9.2 gel
i. HbA HbA (normal human hemoglobin) 20 ul
ii. HbA HbS (heterozygous, or Trait) 20 ul
iii. HbS HbS (sickle cell hemoglobin) 20 ul
Question: Which amino acid residue replaces glutamic acid in sickle cell hemoglobin (HbSHbS)? What is the pI and the migration rate of HbSHbS relative to HbAHbA at pH 9.2 in Part B?
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