Gene Technology Flashcards
What is recombinant DNA technology?
The transfer of fragments of DNA from one organism to another
What are the 5 stages of recombinant technology?
- Isolation
- Insertion
- Transformation
- Identification
- Growth/cloning
What is involved in the first stage - Isolation?
Obtaining fragments of DNA which contain a certain gene (e.g. the one responsible for the production of human insulin). However this is tricky as you must find the gene, and be able to separate it from millions of bases making up the genome
What are the several methods of producing DNA fragments?
- Restriction Endonuclease
- Reverse Transcriptase
- Gene Machine
How does a Restriction Endonuclease remove DNA?
- Recognises DNA and cuts it at a specific sequence of bases, termed a recognition sequence
- This create sticky ends
- These are both palindromic - same sequence on both strands but in reverse
What are ‘sticky ends’?
Single stranded sections of DNA at the end of a double stranded molecule where the bases are exposed/unpaired
How does using a Reverse Transcriptase enzyme cause removal of DNA fragments?
- A piece of mRNA is removed (much easier to find)
2. and is converted in a cell to cDNA (complementary)
Why is using Reverse Transcriptase a better method than using a Restriction Endonuclease?
As the introns are already removed in mRNA, unlike in DNA, and it is much easier to find in the cell as its in high concentrations (translation)
What us the function of DNA Polymerase in Reverse Transcriptase method?
It synthesizes the second DNA strand using the single stranded cDNA as a template - forms a sugar-phosphate backbone as phosphodiester bonds link
Which cell only transcribe the hormone insulin meaning this would be the only cell that the gene could be obtained from?
Pancreatic cells
Pro’s and Con’s of using a gene machine - aka. making your own piece of DNA
+ Made with no introns so prokaryotes can transcribe/translate
+ Any sequence of nucleotides can be produced in a very short period with high accuracy
- Fragments are produced with no sticky ends
- Promotor and terminator regions for the RNA to attach to are also absent at first > need to be added
What are the next stages that can occur after Isolation of the DNA/particular gene has occurred?
- In Vivo Cloning = inside a living cell
2. In Vitro Cloning = outside a living cell
In Vivo cloning is…
This is transferring the DNA fragments into microorganisms which can then be grown and as they reproduce, they’ll pass on a copy of the gene to all new cells
Why would the same Restriction Endonuclease enzyme be used on a DNA sample and the plasmid?
- Cuts DNA at the same recognition sequence
- so get fragments within the gene that are similar, same bit of DNA is isolated
- so that complementary ‘sticky ends’ are formed to allow them to form a recombinant plasmid
Explain the function of a vector in In Vivo cloning?
A vector is used to TRANSPORT DNA into a host cell and ensures that the foreign DNA will be replicated and expressed
What is a plasmid?
A vector which is small, circular DNA found in the cytoplasm of bacteria. It is self-replicating and contains genes that code for ‘marker genes’
Why are plasmids useful in Gene Technology?
Can be easily transferred into other bacteria and contain genes for antibiotic resistance that can be used as a genetic marker
Which enzyme is sued to join the sugar-phosphate backbone of the two strands of DNA and unite them as one within the plasmid, forming recombinant plasmid?
DNA Ligase - ‘seals’ the plasmid
Other products can be formed also (as some plasmids simply re-join and some DNA fragments will join together also) but how are they differentiated by the plasmids which have taken up the DNA fragments?
Detection of the ‘marker genes’ which are added
What is the purpose of the first marker gene added?
Distinguishes the cells that HAVE taken up the plasmid from those who have not.
How does the first marker gene work?
The Transformed cells (that have formed recombinant DNA) are antibiotic resistance so will have survived - so will be grown and cloned on another plate
What is the purpose of the second marker gene?
Distinguishes between the cells that have taken up the Recombinant plasmid from those which have taken up the original plasmid.
How does the second marker gene work?
Foreign DNA is inserted inside the second marker gene ( which the original plasmids will code for) so cells with the recombinant plasmid cannot make that gene product - this is known as REPLICA PLATING
What is the term used when the second marker gene is added?
Replica Plating
When the plasmids are successfully transferred into the bacterial cells, they are said to be…..
TRANSFORMED
How else can a second marker gene be sued?
It can be a gene for resistance to another antibiotic, such as ampicillin where the recombinant plasmid are not resistant. This mean the test would be done on a replica plate, where the colonies are transferred and the ones growing on the second plate (and not the ampicillin plate as they are not resistance to it and would die) are the ones we want !!
Give an example of what can happen when a ‘marker gene’ is added to the desired gene before transformation is attempted?
A gene that codes for a protein that fluoresces/glows in UV light will be easily identified as it would glow green if the desired gene is present
What is the last step of In Vivo cloning?
The bacteria will then be grown on a large scale in industrial fermenters - they will be clones that are each capable of producing the product of the inserted gene
Why chain reaction does In Vitro Cloning (outside the cell) involve?
The PCR = Polymerase Chain Reaction
^identical pieces of DNA are made on large scales
Give some features of In Vitro Cloning?
Automated, rapid and efficient
In the first step of In Vitro Cloning, what pieces need to be added to a thermocycler?
- The DNA fragment to be copied
- DNA Polymerase
- Free nucleotides
- Primers
What is a primer?
A short sequence of single stranded nucleotides, with a specific base sequence. They are complementary to the ends of fragments to be copied and allow DNA Polymerase to bind and start DNA synthesis
Explain the stages in the Polymerase Chain Reaction
In Vitro Cloning
- Temperature at 95 degrees to break H bonds to separate the double stranded DNA
- Temperature reduced to 40 degrees, allowing primers to attach to complementary base sequences
- DNA Polymerase attaches and the temperature is raised to 75 degrees, this is optimum for the enzymes
- Free nucleotides attach by complementary base pairing, making new strands using the old ones as templates
- The process sis repeated and the DNA is doubled each time
Give the order of temperature change during PCR
95 degrees > 40 degrees > 75 degrees
Why is the thermocycler raised to 75 degrees in PCR?
As this is optimum for DNA Polymerase and so the free nucleotides can complementary bind
What type of bond is broken when the DNA is heated to 95 degrees in PCR?
Hydrogen bonds
Why is it necessary in PCR to join primer molecules to the single-stranded DNA before DNA Polymerase is used?
This allows the DNA Polymerase to attach
Starting with a single DNA molecule, the PCR was allowed to go through 6 cycles. How many molecules of DNA would be produced?
The DNA is doubled in quantity each time so:
2x2x2x2x2x2 = 64
(2 to the power of 6)
The original DNA must not be contaminated with any other biological material when carrying out PCR. Suggest why?
- The other biological molecules would contain DNA so
- the other DNA, in the biological molecules, that is undesired may be replicated instead of the desired DNA sample
Explain what is meant by non-coding DNA?
DNA that does not code for proteins
Why AREN’T ribosomes required in PCR?
As translation does not occur, so ribosomes were not needed
Give some examples of how plants can be modified using gene technology techniques
- Adding genes:
> coding for resistance to herbicides
> to develop tolerance to extreme conditions
> to improve the nutrient content of foo
Give some benefits of direct use of genetically modified bacteria
- To increase the quantity of antibiotics and the rate in which they are made
- To produce hormones such as insulin and this avoids killing animals and has fewer side effects on the patient
- To produce enzymes that can be used in the food industry to improve favour
Explain the arguments for and against using genetically modified plants to reduce the population of a particular animal
+ Prevents destruction of crops so prevents spread of disease > economic benefit for farmers
+ More humane than killing them
+ More effective than other methods
+ Reduced use of poisons which may harm other animals as well as the intended ones
However…
- unethical
- Not an instant effect, future generations would be affected the most > passing on of the gene
- Disruption of the food chain
- Causes extinction of that particular animal
What is a potential treatment for genetic diseases, involving altering the genotype?
Gene Therapy
What is the aim of gene therapy?
To supplement the gene, rather than replace it, by adding the normal allele into cells, so it is present alongside the defective one.
How does Gene Therapy work?
The gene would first be inserted into a vector which will deliver the gene into the nucleus of affected body cells following transformation (In Vivo Cloning). The cell will then transcribe the gene into mRNA which would be translated into a protein (by the ribosomes) - healthy, functional proteins are now produced
What does the allele have to be in order for Gene Therapy to work?
A recessive allele - as dominant alleles would overpower the inserted gene
What does Gel Electrophoresis enable scientists to do?
Separate different pieces of DNA on the basis of their length meaning they can then analyze the fragment to produce a genetic fingerprint and locate defective genes using gene probes
What is the process of Gel Electrophoresis?
- DNA samples are first amplified (copied) using PCR, then cut into fragments using a Restriction Enzymes.
- The fragments are placed into wells
- An electric current is passed through the gel in the well and the DNA is attracted to the positive electrode
- The molecules diffuse through the gel: the shorter lengths of DNA move the fastest
Why is DNA attracted to the positive electrode?
Each nucleotide contains a negatively-charged phosphate group
How can the separated fragments on the gel be seen?
- Stain the DNA with a coloured chemical
or - Radioactively label the DNA samples and use an x-ray film to identify the position of the DNA fragments
Q: ‘Suggest how gel electrophoresis separated the proteins obtained from the synapses’
- Depends on size and charge of protein
2. Smaller proteins move the furthest (negatively charged by the R group)
Why would a certain DNA probe only detect a certain allele?
The DNA probe will be a complementary shape to DNA allele A and so will bind to it using hydrogen bonds - acts as a marker gene
On a gel electrophoresis result, why might there only be one band rather than two seen in the same column?
As that person will be homozygous foe the recessive allele or dominant allele so only one band would be shown
Which regions of DNA are used for genetic fingerprinting and why?
The non-coding regions (introns) due to them have bases called Variable Number Tandem Repeats (VNTRs) which have a unique pattern for everyone
What are the five main stages of genetic fingerprinting?
- Extraction > DNA extracted and PCR used to increase quantity of DNA
- Digestion > cut into fragments using Restriction endonuclease, cut close to VNTR areas’
- Separation > Gel Electrophoresis is used, double stranded fragments separated by immersing the gel in alkali
- Hybridisation > Radioactive DNA probes bind to non-coding DNA
- Development > X-ray film placed over sample and a series of bands are seen on the film
Uses of DNA Fingerprinting?
- In Forensic Science
- To determine genetic relationships
- Medical diagnosis
- Plant and animal breeding
What is a DNA probe and what is it used for?
It is a single stranded, short sequence of bases which are complementary to a particular base sequence and can be used to identify whether a certain gene is present in any of the DNA fragments
How are DNA probes produced?
By PCR (using mutant gene templates)
In genetic screening, which part of the DNA is used?
The coding part (extrons) which is opposite to Genetic fingerprinting
