Gene Technology Flashcards
What is gene technology?
All the techniques that can be used to study and alter genes and their function. For example…
- The polymerase chain reaction (PCR)- produces lots of identical copies of a specific gene.
- In vivo gene cloning- also produces lots of copies of a gene
- DNA probes- used to identify specific genes
What are DNA fragments?
The DNA fragment contains the gene you are interested in. There are three methods of obtaining it: - Using reverse transcriptase - Using the polymerase chain reaction - Using restriction endonuclease enzymes
How do you obtain a DNA fragment using reverse transcriptase?
A cell that readily produces the protein is selected.
These have large quantities of the relevant mRNA which is therefore extracted.
Reverse transcriptase is then used to DNA from RNA. This DNA is known as complementary DNA (cDNA) because it made up of the nucleotides that are complementary to the mRNA.
To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene.
Explain why the enzyme is called reverse transcriptase.
Makes DNA from RNA.
How do you obtain a DNA fragment using the Polymerase Chain Reaction (PCR)? 4 steps
Step 1: Reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase.
Step 2: Mixture heated to 95oC to break H-bonds between two strands of DNA then cooled to 50-65oC so primers can bind (anneal) to the strands.
Step 3: Heated to 72oC so DNA polymerase can line up free DNA nucleotides alongside each template strand- specific pairing.
Step 4: Two new copies of the fragment of DNA are formed.
What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want.
What is DNA polymerase?
An enzyme that creates new DNA strands.
What are palindromic sequences of nucleotides?
These sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions). RACECAR
How do you obtain a DNA fragment using restriction endonuclease enzymes?
Restriction endonucleases are enzymes that recognise specific palindromic sequences and cut/ digest the DNA at these places (either side of the DNA fragment you want) via hydrolysis.
Different ones cut at different recognition sequences, because the shape of the recognition sequence is complementary to the active site.
This leaves sticky ends which can be used to bind/ anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
What are sticky ends?
The importance of sticky ends?
Small tails of unpaired bases at each end of a fragment.
- join gene and plasmid together and are specific so only join by complementary base pairing ( must be cut with same restriction enzyme)
- used in invivo gene cloning
Why is using reverse transcriptase good for isolating DNA fragment that has the gene for a desired protein?
- it makes genes without introns, therefore since bacteria are unable to splice out introns it can be expressed in bacteria also
- it makes genes easier to find
What is recombinant DNA
When two DNA strands from different sources join together by sticky ends
What are plasmids?
Circular lengths of DNA found in bacteria
used as a vector to transfer recombinant DNA with the desired gene into a host cell
What are the conditions for transformation?
The plasmid and the host cell/bacteria must be mixed together in a medium containing calcium ions, this combined with changes in temp, make the bacteria permeable hence plasmids can pass through the cell
What is in vitro cloning?
Where the gene copies are made outside of a living organism using PCR.
