How would you approach this problem? You plan to sequence the following DNA by Sanger sequencing. Your reaction includes your sequencing primer (5' is on the left) and template DNA (5' end is on the left), dNTPs, buffer, DNA polymerase and the following fluorescent ddNTPs: red ddGTP, green ddATP and blue ddTTP. Sequencing Primer: CCGCCGGGCCCCAT Template to be Sequenced: GAGCGGCGGGCTGAGTAGCTCGCCGCGGGGATGGGGCCCGGCGGATT
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.
Sanger sequencing, developed by Frederick Sanger in 1977, is a widely used method for determining the sequence of DNA. It involves several key steps:
DNA Template Preparation: Obtain a DNA template containing the region of interest.
Primer Annealing: Design a short DNA primer to bind to the DNA template.
DNA Replication with Modified Nucleotides: Carry out DNA synthesis reactions with regular deoxynucleotides (dNTPs) and modified dideoxynucleotides (ddNTPs), which terminate DNA synthesis.
DNA Synthesis and Fragmentation: As DNA synthesis proceeds, it terminates at ddNTPs, resulting in a set of DNA fragments of varying lengths.
Separation by Electrophoresis: Separate the DNA fragments by size using gel electrophoresis.
Reading the Sequence: Visualize and detect the DNA fragments, and analyze them to determine the sequence of bases.
Sequence Interpretation: Read the chromatogram to determine the DNA sequence based on peak patterns.
Sanger sequencing is known for its accuracy and has been widely used in molecular biology and genetics for sequencing individual genes, detecting genetic mutations, and various other applications. However, it has been largely replaced by high-throughput sequencing methods for large-scale genome sequencing due to their speed and cost-effectiveness.
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