DNA fragments Flashcards
Why are DNA fragments necessary?
For gene technology, copies of gens are needed to work with
Give 3 possible methods of creating DNA fragments
- Conversion of mRNA to cDNA using reverse transcriptase
- Using restriction endonucleases to cut fragments containing the gene from DNA
- Using a gene machine
What is Reverse Transcriptase and its function?
- A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase
- It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins
Describe the process of conversion of mRNA to cDNA using reverse transcriptase
- Obtain cells that produce a lot of the protein and extract mRNA that codes for the protein
- Make a complementary DNA strand (cDNA) using reverse transcriptase and DNA nucleotides
- Hydrolyse the mRNA strand
- Create ds DNA using DNA polymerase and DNA nucleotides
Give an example of using reverse transcriptase [5]
- B-cells from Islets of Langerhans in the Human pancreas
- Extract mature mRNA coding for Insulin
- A single stranded complementary copy of DNA (cDNA)
is formed using reverse transcriptase on the mRNA template - Single stranded cDNA is used to form double stranded DNA using DNA polymerase
- This forms a double stranded copy of the Human Insulin gene
Restriction Enzymes [6]
- Also called restriction endonucleases
- Bacteria contain restriction enzymes in order to protect themselves from invading viruses
- Have highly specific active sites
- Restriction enzymes are used by bacteria to cut up the viral DNA
- Usually cut DNA at specific sites - about 4-8 base pairs long, these are called recognition sites
- This property can be useful in gene technology
Restriction Enzymes - “Blunt Ends”
Some restriction enzymes cut straight across both chains forming blunt ends
Restriction Enzymes - “Sticky Ends” [3]
- Most restriction enzymes make a staggered cut in the two chains, forming sticky ends
- Sticky ends have a strand of single stranded DNA which are complementary to each other
- They will join with another sticky using the enzyme DNA ligase end but only if it has been cut with the same restriction enzyme
Describe the process of using restriction endonucleases to cut fragments containing the gene from DNA
Restriction endonucleases cut DNA at specific palindromic sequences, either side of the desired gene
Describe the process of using a gene machine [5]
- Enter the desired base sequence into a gene machine
- Check for biosafety and biosecurity
- The machine creates small, overlapping nucleotide sequences, oligonucleotides
- Join together to make the gene and replicate using PCR (polymerase chain reaction)
- Once the genes are obtained, they may then have to be modified to add promotor regions, terminator regions, or sticky ends
In vivo cloning involved cloning vast quantities of bacteria.
These bacteria will have specific genes which have been inserted into them. What is the purpose of this?
[3]
- Advantageous Characteristics
- Production of Insulin
- Production of Antibodies
How is in vivo cloning carried out? [4]
- Isolation
- Insertion
- Transformation
- Identification
Isolation [3]
Extract the required gene using one of the following methods:
- Reverse transcriptase
- Restriction endonuclease
- Gene machine
Insertion [6]
- If restriction endonucleases have not been used already, they will be used here
- This creates sticky ends, which allow the gene to be inserted
- Restriction endonuclease cuts gene from genome
- Same type of restriction endonuclease used to cut plasmid / vector
- This means both will have complementary sticky ends, as restriction endonucleases are specific to a particular DNA sequence
- Ligase joins the sticky end of the gene to those of the plasmid / vector
Transformation [3]
- The modified plasmid re-enters the bacterial cell
- There is a around a 1% take up of plasmids, with even less of the bacteria proving to be useful
- A harmless, modified virus could also be used as a vector
Identification [2]
- Plasmids don’t always do what you want them to, therefore, we need ways of identifying the bacteria which have successfully taken the gene/plasmid
- Marker genes are used to identify whether gene has been taken up by the plasmid, and the bacterium has taken up the plasmid
There are several type of Marker Genes [3]
- Antibiotic Resistance Marker Genes
- Fluorescent Markers
- Enzyme Markers
Antibiotic Resistance Marker Genes (replica plating) [6]
- A replica plating plasmid is used which has genes for resistance for 2 different antibiotics
- Plasmid cut at the site of one of these genes and the wanted gene is inserted into this space
- Bacteria is made to grow on agar with 1st (non-cut) antibiotic present
- Any bacteria from this are then transferred to different agar plates with 2nd antibiotic present
- If the plasmid was taken up with gene inserted in 2nd antibiotic bacteria will grow on the 1st plate but not the 2nd
- This will be the useful bacteria
Replica plating is not used much nowadays due to: [3]
- More complicated procedure
- Antibiotic resistance genes may spread and cause resistance in other situations
- Need to look for colonies that haven’t grown on the second plate
Fluorescent Markers [5]
- Plasmid created with GFP (green fluorescing protein) gene
- Desired gene is inserted into GFP gene
- All bacteria will grow, those that glow under UV light have NOT taken up plasmid
- Those that grow but don’t glow contain gene
- This is a much quicker process as it can quickly identify transformed bacteria using UV light
Enzyme Markers [5]
- Plasmid created containing lactase gene
- Lactase turns a substrate from colourless to blue
- Desired gene inserted into lactase gene
- Colonies with gene inserted will not make lactase and will not turn substrate blue
- Colonies without gene inserted will turn substrate blue, and will not be used
What is the difference between in vivo and in vitro cloning?
In vivo = a process that occurs within a living organism
In vitro = a process that occurs in a laboratory vessel
Give 6 differences between in vivo and in vitro cloning [12]
In Vivo:
- Slower
- Uses living cells
- Allows gene expression in a different organism so a specific protein can be made
- Viral vectors have a slight risk of causing an immune response or infection
- Very accurate due to bacterial checking mechanisms
- Low chance of contamination
In Virto
- Very quick
- No cells needed
- Only DNA in sample produced
- Not inserted into living organisms so no immune system issues
- No checking mechanisms
- Contamination common, as any DNA present will be amplified
What does PCR stand for?
Polymerase Chain Reaction
