A student is a lab isolated DNA from a transgenic mouse, a model used to study...

A student is a lab isolated DNA from a transgenic mouse, a model used to study heart failure and wanted to perform the following:

1- Amplify DNA
2- Clone the gene of interest
3- Sequence the sequence of the gene that causes heart failure
What does he need to do for each step

Solutions

Expert Solution

1. Polymerase chain reaction or PCR is commonly employed to amplify the DNA. PCR helps in generating several copies of DNA sequence. For amplification using PCR, the following components are required - (1) DNA template (2) Taq polymerase (enzyme) (3) DNA primers (4) dNTPs (5) buffer solution (6) bi valent cations (magnesium or manganeese). The procedures include - Initialization, denaturation, annealing, extension, final elongation and final hold.

2. Molecular clonning of DNA includes production of identical copies of the gene or DNA of interest. A standard clonning protocol includes the following steps - (1) selection of the clonning vector and host organism (2) Vector DNA preparation (3) creating the typical recombinant DNA (4) Introducing recombinant DNA into host organism (5) Selecting the organism that contains recombinant DNA (6) Clone screening which conatins the DNA insert.

3. Sequencing includes the understanding of the exact nucleotide sequence of the DNA or gene of insert. Sequencing of a DNA may be done using sanger method or maxam -gilbert sequencing.

A typical sanger sequencing is based on the incorporation (selective) of chain terminating dNTPs with the help of DNA polymerase during the process of DNA replication.

The maxam and gilbert method employs the use of radioactive labelling at 5' end of the DNA and chemical treatments produces breaks at certain regions of nucleotide bases. The DNA fragments are seen with the help of electrophoresis and location of each nucleotide is estimated.

gladiator answered 1 year ago

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