Gene Cloning Technology
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What is recombinant DNA?
Fragments of DNA that contain specific genes and have been combined with DNA from other organisms
What is used to cut the DNA?
Restriction endonuclease enzymes
Where is the DNA cut by the restriction endonuclease?
- At a specific sequence of bases
- This sequence is often palindromic
What are the sticky ends in relation to the cut DNA strand?
- The single stranded DNA overhangs
- They are named so because the ends are complementary and will join together by hydrogen bonding
How is MRNA acquired during reverse transcriptase?
It is purified from a cell known to produce the protein of interest
Describe stage 1 of reverse transcriptase
The MRNA is purified from a cell known to produce the protein of interest
Describe stage 2 of reverse transcriptase
The MRNA is mixed with DNA nucleotides and the enzyme reverse transcriptase
Describe stage 3 of reverse transcriptase
- The DNA nucleotides join to the complementary mRNA bases
- The enzyme joins the DNA nucleotides together forming a complementary strand of DNA known as cDNA (complementary DNA)
Describe stage 4 of reverse transcriptase
- The cDNA is mixed with more DNA nucleotides and the enzyme DNA Polymerase
- The cDNA is then used as a template to produce a double stranded molecule of DNA
How is reverse transcriptase useful for cloning eukaryotic genes?
The mRNA will lack introns meaning the gene can be expressed in bacteria
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