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Reverse Transcriptase and DNA Synthesis Techniques

The document explains the process of creating DNA fragments from mRNA using reverse transcriptase, which produces cDNA that is intron-free. It also discusses restriction enzymes that cut DNA at specific locations, creating either blunt or sticky ends for DNA joining. Additionally, it describes the use of a gene machine to design and synthesize DNA sequences based on amino acid analysis, ensuring biosafety and biosecurity, followed by PCR amplification to produce double-stranded DNA.

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3 views2 pages

Reverse Transcriptase and DNA Synthesis Techniques

The document explains the process of creating DNA fragments from mRNA using reverse transcriptase, which produces cDNA that is intron-free. It also discusses restriction enzymes that cut DNA at specific locations, creating either blunt or sticky ends for DNA joining. Additionally, it describes the use of a gene machine to design and synthesize DNA sequences based on amino acid analysis, ensuring biosafety and biosecurity, followed by PCR amplification to produce double-stranded DNA.

Uploaded by

schreoshimkhan
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

Reverse transcriptase - creates DNA fragments from mRNA using the enzyme reverse

transcriptase

This enzyme naturally occurs in viruses, such as HIV, and it makes DNA copies from mRNA

A cell that naturally produces the protein of interest is selected.

These cells should have large amounts of mRNA for the protein.

The reverse transcriptase enzyme joins DNA nucleotides with complementary bases to the
mRNA sequence.

Single-stranded DNA is made (cDNA).

To make this DNA fragment double-stranded, the enzyme DNA polymerase is used.

The cDNA is intron free because it is based on the mRNA template.

Restriction enzymes

Restriction endonucleases are enzymes that cut up DNA. They naturally occur in bacteria as
a defence mechanism.

There are many restriction enzymes that have an active site complementary in shape to a
range of different DNA base sequences, described as recognition sequences.

Therefore each enzyme cuts the DNA at a specific location.

Some enzymes cut at the same location in the double-strand and create a blunt end, other
enzymes cut to created staggered ends and exposed DNA bases.

The exposed staggered ends are palindromic and referred to as ‘sticky ends’ because they
have the ability to join DNA with complementary base pairs.
Gene machine

DNA fragments can be created in a lab using a computerised machine.

Scientists first examine the protein of interest to identify the amino acid sequence, and from
that work out what the mRNA and DNA sequence would be.

The DNA sequence is entered into the computer, which checks for biosafety and biosecurity
that the DNA being created is safe and ethical to produce.

The computer can create small sections of overlapping single strands of nucleotides that
make up the gene, called oligonucleotides.

The oligonucleotides can then be joined to create the DNA for the entire gene.

PCR is used to amplify the quantity and to make the double-strand.

This process is very quick, accurate and makes intron free DNA so can be transcribed in
prokaryotic cells.

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