8.4 Gene technologies Flashcards
What is recombinant DNA technology
The transfer of fragments of DNA from one organism, or species, to another
What is the name of the organism that receives the fragment of DNA
Recipient
What 2 reasons mean the recipient can translate the DNA within the cell
- Genetic code is universal
- Transcription and translation mechanisms are universal
What is the recipient organism said to be after receiving the DNA fragments
Transgenic
What 2 fields may recombinant DNA technologies be beneficial
- Medicine
- Agriculture
Define donor DNA
Gene that is isolated for insertion
Define plasmids
Circular loops of DNA can be used as vector
What is meant by the word vector in terms of recombinant DNA technology
Carries the DNA into recipient
Define restriction endonucleases
Enzymes that cut DNA at specific restriction sites
Define DNA ligases
Enzymes that join sections of DNA together
Define sticky ends
2 ends of ‘cut’ DNA segments that have unpaired
Define recombinant DNA
DNA which is formed when a piece of foreign DNA is incorporated into the plasmid from a bacterium
Define reverse transciptase
Enzymes used to synthesise DNA from mRNA in specific cells
Define clone
A population of genetically identical cells or organisms
What happens in the isolation part of the DNA technology
Isolating DNA fragment that contains the gene of desired protein
What are the 3 steps when isolating the desired gene
- Locate the gene, in cells that naturally produce the protein
- Identify gene locus using a gene probe
- Isolate gene from DNA
What organism does restriction endonucleases naturally occur in
Bacteria
What are the names of the sites where the restriction endonucleases cut the DNA
At specific restriction sites/ recognition sites
What bonds do restriction endonucleases hydrolyse
Phosphodiester bonds
Recognition sites are always _________
Palindromic
What does palindromic mean
Read the same forward and backward
What do restriction endonucleases do
Cut double-stranded DNA into fragments
What are the steps involved in using reverses transcriptase to isolate the desired gene
- Extract mRNA that codes for the gene from a cell that naturally produces the protein
- Mix the mRNA with reverse transcriptase, so complementary DNA (cDNA) is formed
- Add the single-stranded DNA strand with DNA polymerase
- A double-stranded DNA will form
What are the advantages of using reverse transcriptase to isolate genes compared to restriction endonucleases
- There are far more mRNA molecules that carry the desired gene compared to DNA
- mRNA is in the cytoplasm whereas DNA is in the nucleoplasm so fewer membrane to pass
- No introns in mRNA, so can be put into bacteria cells
Why is it beneficial to have no introns in the gene that’s transferred into bacteria
Since bacteria have no introns so don’t know what to do with it
What is needed for the gene machine to work
The primary structure of the protein
Why does the primary structure of the protein need to be known to use the gene machine
- Identify the amino acid sequence
- Then determine the mRNA codons
- Then determine the complementary DNA triplets
- The determine the base sequence needed for the gene machine
What does the gene machine produce
Short DNA fragments
What is the name of the short DNA fragments that the gene machine produces
Oligo-nucleotides
What is the name of the enzyme that joins the oligo-nucleotide together in the gene machine
DNA ligase
What are the benefits of the gene machine
- Intron free
- Most accurate
- Fastest
Where the DNA of 2 different organisms is combined, the product is known as
Recombinant
One method of producing DNA fragments is to make DNA from RNA using an enzyme called
Reverse transcriptase
Once restriction endonucleases have cut the DNA, it leaves 2 types of ends, name the 2 types
- Sticky ends
- Blunt ends
During the insertion process of gene technology, what is inserted into one end of the gene which enables transcription factors to bind
Promoter region
During the insertion process of gene technology, what is inserted into the other end of the gene which releases RNA polymerase to end transcription
Terminator region
When inserting gene into vector, what is important about the restriction endonucleases that are used
They must be the same, the same enzyme that cut the donor DNA into small fragments must cut the plasmid too
Why must the vector and the donor DNA be cut with the same restriction endonucleases
So the sticky ends are complementary on the gene and the plasmid
What is the role of DNA ligase in insertion process
Splice the gene into the vector
When using a plasmid as a vector, what may happen after the plasmid has been cut open
The plasmid may bind to itself again without the donor DNA within
How are plasmids reintroduced
By mixing plasmids with bacteria cells, so bacteria can take plasmids up
What is the general term for the things that are used to identify bacteria with genes
Genetic markers
What are genetic markers
Genes that are easily identifiable
What are the 3 main genetic markers
- Genes that result in antibiotic resistance
- Genes that code for fluorescent proteins
- Genes that code for enzymes whose action can be identified
How is the donor DNA spliced into a plasmid with antibiotic resistance to be used as a genetic marker
Cut the plasmid with the 2 resistant genes, in the centre of one of the resistant genes and insert the fragment of donor DNA inside.
How does the donor DNA being inserted into resistant region on a plasmid act as a genetic marker
- The bacteria that has the recombinant plasmid won’t be resistant to the antibiotic where the donor DNA was added to
What are the 3 possible option of bacteria after plasmid have had fragments of DNA added and mixed with the bacteria
- Bacteria with no plasmids
- Bacteria with recombinant plasmids
- Bacteria with original plasmids
How can scientists identify which bacteria has which type of plasmid
Replica plating
Where is the gene inserted in the vector, when fluorescent markers are used
In the middle of the GFP gene
Are bacteria with recombinant plasmid that have fluorescent markers, fluorescent or not
Not fluorescent
What does lactase do
Turn colourless substrate of lactose into a blue product
How are enzyme markers used to identify bacteria with recombinant plasmids
Genes are added to the centre of lactase, so bacteria with recombinant plasmids are white
What happens in the culturing process of DNA technology
- Remove the identified bacteria
- Add to tank for fermentation
- Where the bacteria replicate by binary fission which produces genetically identical bacteria all containing recombinant plasmids
What does in vivo mean
Inside of living organism
What does in vitro mean
Outside of living organism
What is the method when cloning DNA in vitro
PCR machine
What is the method when cloning DNA in vivo
Using bacteria cells to clone DNA
What does PCR stand for
Polymerase chain reaction
What is the role of PCR
DNA amplification
What does the PCR machine do to the number of DNA molecules
Increase the number exponentially (double the number per unit of time)
What is added to the PCR machine (4 things)
- DNA nucleotides
- The gene
- Primers
- DNA polymerase
What type of DNA polymerase is used in the PCR machine
Taq
What are primers
Short single-stranded DNA
What are the roles of primers in PCR machine (2 roles)
Primers are complementary to the bases at the start of the gene we wish to clone, and primers signal DNA polymerase to start synthesising bonds
After everything is added to the PCR machine, what temperature is the machine heated to
95 degrees celsius
Why is the PCR machine heated once everything is added to the machine
High temperature breaks the hydrogen bonds, which unzips the DNA, so both strands can act as template
After the PCR machine is heated to 95 degrees, what temperature is the machine then cooled to
55 degrees celsius
Why is the PCR machine cooled to 55 degrees
Since this is the temperature that the primers complementary base pair (anneal) to DNA strand
What is the biological word for when primers complementary base pair with DNA strand
Anneal
What is the advantage of primers annealing with DNA strand
To stop DNA from rejoining together
After the PCR machine is cooled, what temperature is the machine then heated too
72 degrees celsius
Why is the PCR machine heated to 72 degrees
Since this is the optimum temperature for Taq DNA polymerase
What are the 5 limitations of PCR
- Contamination to solution
- Error rate
- Size of DNA fragment that is copied
- PCR is sensitive to inhibitors
- Limit to amplification
Why is contamination a limitation to PCR
Any DNA in machine is amplified
Why is error rate a limitation to PCR
Taq DNA polymerase cannot proof read mutation in DNA replication so errors accumulate
Why is size of DNA fragments a limitation to PCR
PCR machine cannot read anymore that 3000 bases
Why is limit to amplification a limitation to PCR
- Exponential growth only occurs for roughly 20 cycles, since enzymes begin to denature, concentration of nucleotides decrease
What is the comparative statement:
In vivo cloning culturing techniques are required
In vitro cloning no culturing techniques are required
What is the comparative statement:
In vivo cloning once in bacteria the DNA will automatically be copied
In vitro cloning, requires correct primers to be present for copying to occur
What is the comparative statement:
In vivo cloning there’s almost no risk of contamination
In vitro cloning any contaminated DNA will also be amplified
What is the comparative statement:
In vivo cloning DNA polymerase has ‘proof reading function’
In vitro cloning DNA polymerase does not have ‘proof reading function’
What is the comparative statement:
Vivo cloning is very accurate
Vitro cloning errors accumulate
What is the comparative statement:
In vivo cloning transformed bacteria can synthesise gene product
In vitro cloning genes must be inserted into cells to synthesise gene products
What is the comparative statement:
Vivo cloning is relatively slow
Vitro cloning is extremely rapid
What is the comparative statement:
In vivo cloning specific genes are isolated
In vitro cloning whole or broken down DNA can be copied
What is the comparative statement:
In vivo cloning copying can be effective up to 3 million base pairs
In vitro cloning copying can be effective up to 3000 base pairs
