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.
What is in vivo cloning?
Where the gene copies are made within a living organism. As the organism grows and divides, it replicates its DNA, creating multiple copies of the gene.
What are the three steps to in vivo cloning?
- Making recombinant DNA
- Transforming cells
- Identifying transformed cells
Part 1: Making recombinant DNA
The vector DNA is isolated.
The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment. This means that the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment containing the gene.
DNA ligase joins the sticky ends.
Recombinant DNA formed
What is a vector?
Something that’s used to transfer DNA into a cell.
They can be plasmids (small circular molecules of DNA) or bacteriophages (viruses that infect bacteria).
Part 2: Transforming cells
The vector with the recombinant DNA is used to transfer the gene into host cells.
Plasmid vector is used- host cells placed in ice cold calcium chloride solution to make their cell walls more permeable.
The plasmids are added and the mixture is heat shocked (heated to around 42 degrees for 1-2 minutes), which encourages the cells to take in the plasmids.
Part 3: Identifying transformed cells
Marker genes can be inserted into vectors at the same time as the gene to be cloned.
Host cells are grown on agar plates and each cell divides and replicated its DNA, creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene.
The marker gene can code for antibiotic resistance- if the host cells are grown on agar plates containing the specific antibiotic only transformed cells that have the marker gene will survive and grow. Marker genes can also code for fluorescence when the agar plate is placed under a UV light only transformed cells will fluoresce.
Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene.
COMPARISON: mRNA
1) pentose sugar
2) bases
3) shape
4) hydrogen bonding
ribose
AUGC
single-stranded helix
no H bonding
COMPARISON: tRNA
1) pentose sugar
2) bases
3) shape
4) hydrogen bonding
ribose
AUGC
single-stranded helix folded into clover shape and held by H bonds between complementary base pairs
COMPARISON: DNA
1) pentose sugar
2) bases
3) shape
4) hydrogen bonding
deoxyribose
ATGC
double-stranded helix twisted and helpd by H bonds (2x polynucleotide chains)
Advantages of in vitro gene cloning.
Very fast.
Does not require living cells.
Advantages of in vivo gene cloning.
Useful where we wish to introduce a gene into another organism as it involves vectors.
No risk of contamination.
