A possible forward primer to be designed is a primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located. In order to clone the PCR product generated using these primers into pET-28a, extra nucleotides must be added to the 5' ends of the primers. These extra nucleotides will contain the recognition sites for the restriction enzymes that are used for cloning. When the DNA produced by PCR using these primers is treated with the restriction enzymes, this will generate sticky ends at the ends of the PCR product. If pET-28a is digested with the same restriction enzymes, the sticky ends of the PCR product will bind to the sticky ends on the digested pET-28a, allowing the two DNA molecules to be connected together by DNA ligase. Which primer (forward or reverse) should contain the SacI recognition site, and which primer (forward or reverse) should contain the HindIII cleavage site? Why? [3 – 5 sentences suggested] Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.
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.
A possible forward primer to be designed is a primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located.
In order to clone the PCR product generated using these primers into pET-28a, extra
Which primer (forward or reverse) should contain the SacI recognition site, and which primer (forward or reverse) should contain the HindIII cleavage site? Why? [3 – 5 sentences suggested]
Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.
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