3.8.4.1 Recombinant DNA Technology Flashcards
What is recombinant DNA technology?
A form of genetic modification
What does transgenic mean?
Genetically modified
What is isolation?
The isolation of the DNA fragments that have the gene for the desired protein
What is insertion?
The insertion of the DNA fragment into a vector
What is transformation?
The transfer of DNA into suitable cells
What is identification?
The identification of the host cells that have successfully taken up the gene by use of the gene markers
What are the steps of recombinant gene technology?
Isolation Insertion Transformation Identification Growth / cloning
What are the methods of producing gene fragments?
Conversion of mRNA to cDNA using reverse transcriptase
Using restriction endonucleases to cut fragments containing the desired gene from DNA
Creating the gene in a gene machine
What do endonucleases do?
They break up nucleotides
What is a blunt end?
When DNA is cut straight through the double strand without seperating bases
What is a sticky end?
When DNA is cut in a staggered fashion This usually happens when the code on one strand is pallindromic e.g. A G C G C T T C G C G A
What is a DNA fragment?
The DNA segment between the two cuts
What are the sticky ends on the fragment complimemtary to?
The sticky ends on the plasmid
What is the proces of using reverse transcriptase?
Start with the mRNA code
The reverse transcriptase forms a complimentary strand of DNA, called cDNA
Single-stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
Double-stranded DNA is formed on the template of the cDNA using DNA polymerase
What does reverse transcriptase produce?
DNA with no noncoding DNA
What is the process of using restriction endonucleases?
Each type of these enzymes cuts a DNA double strand at a specific sequence of bases
This leaves two blunt ends
Others cut in a staggard fashion
What do restriction endonucleases produce?
cDNA (noncoding DNA)
What is the process of using a ‘gene machine’?
You need to work out the amino acid sequence first
This means you should know the mRNA codons and therefore the DNA triplets
The desired sequence of nucleotide bases is fed into a computer
The computer designs oligonucleotides
Oligonucleotides are short pieces of single-stranded DNA which are joined together to make a gene
It is quicker to create smaller strands then join everything together, base by base
The polymerase chain reaction constructs the complementary strand
What is the process of insertion?
DNA ligase joins the DNA fragment and vector (ligation)
This forms recombinant DNA
A promotor region is a section of DNA at the end of the gene where DNA polymerase binds to
The nucleotide bases of the promotor attach both RNA polymerase and transcription factors, and so begin the process of transcription
Attaching the promotor is essential for transcription
A terminator must also be added to stop transcription at the appropriate point
At this point you have a recombinant plasmid
If you use restriction enzymes, introns are present and splicing is required
What is a vector?
A vector carrier DNA into a host cell
What does using a gene machine produce?
Coding DNA
What is the process of transformation?
First increase the permeability of the membranes, e.g. use calcium ions for bacteria
Keep cold to reduce metabolic activity
Once its exposed quickly raise the temperature to 42 (heat shock)
This causes the plasmid to be quickly expadited into the bacteria
This encourages the movement of the vector into the host cell
What are the problems that can occur during during insertion?
The DNA fragments can join up with each other because it is looking for complimentary pairs
Sometimes plasmids close up before the gene has been inserted because the sticky ends are complementary
Sometimes two plasmids join
What are the advantages of recombinant DNA technology?
GMO plants produce plant organs which can then be harvested
Replacing defective genes can be used to cure genetic disorders
GMO crops can survive harsher conditions
What are the risks of recombinant DNA technology?
Can a company patent a gene and own DNA?
Could a new disease be created accidentally?
People could change their genetic makeup (eugenetics)
What are DNA probes?
Short single strands of DNA which contain a DNA marker
What are radioactively labelled probes?
Made up of nucleotides with the isotope 32P
Identified using an x-ray film
What are fluorescently labelled probes?
Probes which emit light
What is the process of DNA hybridisation?
Seperate the two strands of DNA
Add complimentary DNA from someone else to see if they’re complimentary
What is hybrid DNA?
A form of recombinant DNA where the complimentary parts of the strand anneal
What is recombinant DNA?
DNA from two different source
What does the annealing of probes do?
It confirms the base sequences
What can DNA be split up by?
Heat or DNA helicase
What is the function of PCR?
It amplifies your sample of DNA
How long is the DNA sample in PCR?
An entire genome or a fragment
What does PCR stand for?
Polymerase chain reaction
What is required for PCR in terms of DNA polymerase?
DNA ploymerase will denature at higher temperatures
What is required for PCR? (without explanation)
DNA polymerase pH buffer Thermal cycle Primers Free nucleotides
What is required for PCR in terms of the thermal cycler?
All ingredients are put into a themal cycler which changes the temperature
What are primers?
Short sequences of nucleotides that have a set of bases which are complimentary to the DNA fragments
What are the three steps of PCR? (no explanation)
Denaturing
Annealing
Extension
This continues in a cycle
What is the process of denaturing in PCR?
95 degrees celcius
The strands seperate
Hydrogen bonds break
DNA fragments, primers and taq polymerase are added
What is the process of annealing in PCR?
Cooling down to 55 degrees celcius Primers attach to each strand of DNA Each primer covers 2+ bases Primers prevent the strand from rejoining Primers anneal to the DNA This helps the taq polymerase to bind
What is the process of extension in PCR?
Heated to 72 degrees celcius Also called elongation End up with two double strands of DNA Free nucleotides attach Taq polymerase joins the free nucleotides
What antibiotics are neccesary for identification? (no explanation)
Ampicillin, Tetracycline
What are the two types of identification?
Antibiotic resistance or fluorescence
What is replica plating?
Sterile velvet is placed onto the first plate to make a copy of the colonies
This is then placed onto another plate
The plates are then compared to see which colonies were killed by Ampicillin so carry the desired gene
All done in sterile conditions
What is the stage of growth?
Colonies grown in nutrient growth
Rinse with Ampicillin at the end to kill remaining bacteria
What role does Ampicillin play in identification?
The gene for ampicillin resistance is disrupted by the inserted gene fragment, any plasmid killed by ampicillin has had the DNA fragment succesfully inserted
What role does Tetracycline play in identification?
The gene for tetracycline is not disrupted by the DNA fragment, if the bacteria doesn’t have a plasmid it will be killed by tetracycline
How is fluorescene used for recombinant DNA identification?
Add a fluorescent gene to the DNA fragment
Insert the whole thing into the vector
If the bacterium is fluorescent, the desired gene has been inserted
What should you do when attempting to use fluorescence identification?
Use a large sample of DNA so that there is still a large amount of DNA left after several attempts
What is the difference between in vivo and in vitro cloning?
In vivo: the bacteria do it, the process of isolation + insertion + transformation + identification + growth
In vitro: a machine does it, polymerase chain reaction
