General Flashcards
Define biotechnology.
The use of an organism, or component of an organism or other biological system, to make a product of process.
Define molecular biology.
The study of the molecular basis of the processes of DNA replication and the central dogma.
What is DNA technology?
Applying DNA-based techniques for the manipulation of genes and genetic material at the molecular level.
What is biotechnology used to discover - name some.
Evolution, gene expression, growth and development, genetic diseases, roles of proteins.
Why is molecular genetics difficult ?
DNA molecules are very long and carry many genes. Only a small portion of the DNA molecule will code for genes and it is difficult to find where a gene begins and ends in a long nucleotide sequence.
What is the overall purpose of gene cloning?
Creates multiple copies of the desired gene.
What must be present for gene cloning to occur?
Cloning vectors.
What is rDNA?
Recombinant DNA - DNA recombines from 2 different sources.
What are the 2 possible aims of gene cloning?
To produce a protein product for further research or practical use.
OR
To produce multiple copies of a gene.
Why is gene cloning used to produce multiple copies of a gene?
Sequencing analysis, to transform an organism with the gene.
Name methods of gene cloning.
PCR - polymerase chain reaction.
Using recombinant gene technology.
How overall does the process of genetic cloning work?
Isolation of plasmid DNA and DNA containing gene of interest.
Gene of interest inserted into plasmid.
Recombinant DNA formed.
Recombinant Plasmid inserted into bacterial cell.
Cells clones with gene of interest.
Make copies of genes formed.
Name the 2 most common DNA vectors.
E.coli
Bacteriophage gamma vectors
Why don’t we use gene cloning to produce carbohydrates?
Genes code for proteins not sugars.
What is the benefit of using E.coli as a DNA vector in gene cloning?
It divides rapidly (every 30 minutes) and so the desired gene is amplified rapidly.
What is the purpose of polymerase chain reaction (PCR)?
Used to clone and amplify a specific section of DNA.
What is meant by PCR amplifying DNA?
Copying it many times.
What cloning vectors and bacterial cells does the polymerase chain reaction require?
None
Where is DNA amplified by the polymerase chain reaction?
In vitro
What is the purpose of gel electrophoresis?
Used to sort a mixture of DNA into molecular bands. Each band contains DNA molecules of the same base-pair length.
What method is used to visualise and analyse produces of the polymerase chain reaction?
Gel electrophoresis
What method is used to visualise and analyse restriction fragments of DNA?
Gel electrophoresis.
What are restriction fragments of DNA?
Sections of DNA which are produced as a result of a restriction endonuclease cutting the DNA.
What are the characteristics of DNA molecules within each band of gel electrophoresis?
All have the same base-pair length.
Define proteome.
Every protein that the gene is capable of making.
What is DNA profiling?
Using DNA to distinguish between individuals.
Name some practical uses of DNA profiling.
Paternity identification
Victim identification
Suspect identification
What section of the DNA profile identifies different individuals and why?
Short tandem repeats?
They are different in every individual and have varying number of repeat sequences?
How do the short tandem repeats of a DNA profile change in every individual?
They contain a different number of repetitive sequences.
What are polymorphic regions of DNA?
Varying regions of DNA in the non-coding sections of the genome. Site of heterozygosity for any sequence.
What type of cell was ‘Dolly the sheep’ cloned from?
Adult somatic cell - udder cell of an ewe
Explain how the cloning of ‘Dolly the sheep’ proved that all cells of an individual share a common genetic blueprint.
Donor cell = udder cell of a white faced ewe.
Surrogate mother = black-faced ewe.
Dolly was an exactas genetic duplicate of the donor ewe and was un-related to the surrogate.
What were the main findings from cloning ‘Dolly the sheep’ ?
All cells of an individual share a common genetic blueprint.
Outline reproductive cloning.
The nucleus from a donor egg cell is grown in culture to produce an early embryo. This is then implanted into the surrogate mother. A clone of he donor cell is born.
Outline therapeutic cloning.
The nucleus from a donor egg cell is grown in culture to produce an early embryo. The embryonic stem cells are removed from the early embryo and grown in culture. This induces stem cells to form specialised cells for therapeutic uses.
What are some of the main purposes of cloning animals?
Herd improvement, farming improvements, saving endangered species.
What are adult somatic cells?
Adult stem cells which are undifferentiated and divide to replenish dying cells or damaged tissues.
What are transgenic organisms?
Organisms whose genomes carry genes from one species to another species.
Give an example of transgenic organisms.
Genetically modifies crops
Give a use of transgenic organisms.
To produce better livestock
What are “pharm’ animals?
Animals designed to be pharmaceutical factories. They produce proteins for medical use.
What does ‘GM’ stand for in terms of ‘GM crops’ ?
Genetically modified
Outline how genetic modification occurs in humans.
The cloned gene for the desired human protein is injected into the nuclei of the in vitro fertilised eggs.
The genetically modified eggs are implanted in the surrogate mother.
What is meant by ‘in vitro’?
A process outside of the living organism. (Test tube/culture dish).
How are most genetically modified proteins secreted in animals?
Via the milk
Are genetically modified animals and plants part of the food chain?
Animals - no
Plants - yes
What is the purpose of having transgenic crop plants?
They have desirable traits such as delayed ripening and pest resistance.
What were the main aims of the human genome.
To map the entire human genome and determine the complete nucleotide sequence of the DNA of each human chromosome.
Also used to establish the evolutionary relationships between species.
How many genes were found in the human genome?
About 21000
Compare the human genome to the quantity of genes it has.
The human genome is much larger than other species but has relatively few genes for its size.
Who made/ theorised the DNA sequencing machine.
Fredrick Sanger
What is the purpose of DNA sequencing machines?
Used to form clones copies of short fragments of DNA.
What methods are used in DNA sequencing machines?
DNA labelling
DNA synthesis - in vitro including special chain-termination nucleotides.
High resolution gel electrophoresis.
Give an example of ‘high-throughput’ technology.
Sequence by synthesis
What is the main purpose of ‘sequence by synthesis’.
Copy fragments of DNA
Outline the process of ‘sequence by synthesis’.
A specific strand of each fragment of DNA is immobilised and the complimentary strand is synthesised one nucleotide at a time.
A chemical technique enables electronic monitors to identify in real time, which of the 4 nucleotide bases is added.
What are the bonfires of using ‘third-generation’ sequencing over ‘sequence by synthesis?
It is faster and cheaper
How is a DNA sequence identified using nano pores?
Long DNA molecules move through tiny nano pores. Each type of base is identified by the way it interrupts the chemical Current passing through the pore.
What methods are used to detect pathogens which may cause hereditary or regular genetic modifications?
PCR - polymerase chain reaction
Probes
How do human diseases arise from protein defects?
Disease-causing alleles cause a dysfunctional protein or no protein to be coded for.
What is incomplete dominance ?
One allele for a specific trait is not completely expressed over its allele pair. This results in a third phenotype in which the expressed phenotype is that of both alleles.
Name a form of intermediate inheritance for genetic diseases.
Incomplete dominance
Outline some uses of Gene cloning.
Medicine, Agriculture, Pharmaceuticals
What are the 2 sections joined to form recombinant DNA?
Vector (plasmid) and target segment of DNA.
How are the plasmid and target DNA joined to form recombinant DNA?
DNA ligase joins the 2 segments by forming covalent phosphodiester bonds. Phosphate on the 5’ end of DNA is joined to the hydroxyl group on the 3’ end of the other DNA section. Creates sugar-phosphate backbone.
Uses ATP
What are the 3 required characteristics of vectors?
Ability to replicate independently of the host chromosome.
A detectable genetic marker able to select for host cells containing the vector and any attached DNA.
Single site for 1 or more restriction enzymes, allowing DNA fragments of interest to be inserted at a defined point in the vector.
What is a plasmid?
A small circular, single-stranded molecule of DNA which exists in bacterial cells.
Why are plasmids not required for the replication and survive of bacteria?
They contain very few genes
Why can’t bacterial plasmids contain outside the host cell?
They don’t have a protective protein coat.
Briefly, how do we know that our specific gene has been transformed?
The genes will survive in nutrient agar due to Ampr expression.
Who developed the first versatile cloning vector pBR322?
Herb Boyer
What is meant by the term ‘vector’ in terms of plasmids?
The use of plasmids as vehicles to transport DNA from test tube to host cells.
What type of E.Coli cells from ampicillin resistant colonies?
All which have been transformed.
Describe the plasmid and DNA present in each cell of the same colony produced by gene cloning.
Each cell will have the same plasmid and DaN.
Give some uses of recombinant DNA.
Isolation of genes to determine nucleotide sequences.
Identify and analyse specific DNA sequences.
PRotein/ RNA investigation.
Identification of mutations.
Engineering of organisms for specific uses.
When were endonuclease enzymes discovered?
Late 60s
What are endonculeases?
Restriction enzymes thatch DNA at a specific site and protect the DNA from viruses and other foreign sources of DNA.
What is restriction in terms of molecular genetics?
The process of cutting foreign DNA.
What do restriction enzymes recognise?
Specific nucleic acid sequences known as restriction sites.
What are restriction sites?
Specific sequences of nucleic acids which are recognised and cleaved by restriction enzymes.
What feature is usually true in restriction sites.
They are usually palindromes.
What are palindromes?
Sequences of DNA which read the same forwards and backwards.
(madam , race car)
How do bacterial cells protect their own DNA from restriction?
Adding methyl groups to the same restriction site that will be cleaved on the plasmid and isolated DNA gene on its own genome.
What was the first endonuclease characterised?
EcoR1
What do restriction enzymes do to ‘cut’ DNA?
They hydrolyse phosphodiester bonds.
What are sticky ends?
When the restriction enzyme cuts the DNA in a staggered way.
What is it called when restriction enzymes cut the DNA in a straight line?
Blunt ends
What are blunt ends?
When the restriction enzyme cuts the DNA to have straight ends.
Why are sticky ends beneficial in gene cloning?
They produce recombinant DNA more easily than blunt ends because the sticky ends of the vector plasmid and DNA are able to complimentary base pair and then use DNA ligase to seal phosphodiester bonds between the 2 molecules.
This forms rDNA.
Why was E.Coli K-12 a popular bacteria used in gene cloning?
Easy to grow.
Open and responsive to metabolic studies.
A good host for the propagation of recombinant DNA molecules.
What year was E.Coli K-12 first isolated in?
1921
What gene is E.Coli K-12 mainly used to study?
Operons (lac).
What are the important genesections carried on the pUC19 plasmid?
Ampr - ampicillin resistance.
LacZ - Encodes for beta galactoside enzyme.
Outline the process of using nutrients agar to find transformed genes.
The bacteria are plated out onto nutrient agar containing ampicillin and X-Gal sugar.
Every viable bacteria forms a colony of clones.
Amplicillin ensures that only plasmid-containing cells grow. X-Gal selects recombinant plasmids.
X-gal is hydrolysed by beta galactoside to yield a blue produce.
Colonies containing intact plasmids will appear blue.
Plasmids with the inserted human gene won’t produce beta galactoside and will be white.
The desired white colonies can then be isolated directly from the agar plate.
What colour will the desired colonies be on an agar plate after gene cloning?
White
What is the purpose of ampicillin being present on the agar plate used to identify transformed genes?
Ensures only plasmid-containing cells grow.
What is the purpose of X-Gal being present on the agar plates used to identify transformed genes?
Is key for selecting cells with recombinant plasmids.
What colour will colonies with intact plasmids appear on nutrient agar identification?
Blue
What colour will colonies containing the desired gene appear on nutrient agar plates?
White
Why do the desired genes of genetic cloning need to be further identified after nutrient agar identification?
This will visualise clones of different DNA fragments not just the specific gene of interest.
What is used to identify the specific colony of the gene desired after nutrient agar has been carried out?
Nucleic acid probes
What are Nucleic acid probes used for in genetic cloning?
To distinguish between the colony containing the desired gene and the other irrelevant colony fragments.
What is the process called when a plasmid is introduced into a bacterial cell?
Transformation
Clone definition
To make an exact genetic copy
What genes are generally contained in plasmids and are beneficial to bacteria?
Antibiotic resistance genes
Why do only bacteria containing plasmids survive on agar nutrient plates?
The bacteria with plasmids will contain antibiotic resistant genes and will therefore survive to produce colonies.
However, some bacteria will have no plasmids and will therefore be killed by antibiotics.
How are bacteria encouraged to take up plasmids during transformation?
They are given a shock such as sudden exposure to high temperatures.
Why does heat shock make bacteria take up plasmids?
It changes the fluidity of the membrane and causes pores to form. This makes it easier for the DNA to enter the cell.
What allows human genes to be expressed in bacteria?
Humans and bacteria share the same DNA and RNA.
What will prevent human genes being transcribed and translated in bacteria and why?
If introns are present , transcription and translation won’t be possible because bacteria cannot splice RNA transcripts.
What type of DNA is produced when the introns of the DNA are removed to allow gene cloning in bacteria?
cDNA - complementary
How are the proteins produced by genetically cloned DNA released from bacterial cells?
Bacterial cells are split open.
What method is used to purify the insulin produced by genetic cloning in bacteria?
Column purification
How is the insulin produced by genetic cloning made to be in its active form?
Produced in it’s inactive form by bacteria.
Disulfide bonds must form between separate chains. The chains are then split and only held together by disulphide bridges.
What is the purpose of tissue plasminogen activator in the body?
Used to treat strokes and prevent blood clots.
What type of protein is tissue plasminogen activator?
Recombinant protein
What is the purpose of gene therapy?
To provide a normal copy of the gene to a patient who has a faulty or lacking version of a specific gene.
How does gene therapy help people with cystic fibrosis?
Reduces the rate of lung deterioration.
Where is insulin produced?
Pancreas
What is Type 1 diabetes?
The cells of the pancreas that produce insulin are damaged or destroyed so insufficient insulin is produced.
Insulin from which animals can be used to treat type 1 diabetes?
Humans, pigs cows
What is the purpose of southern blotting in genetics?
To verify the presence or absence of a specific nucleotide sequence in DNA from difference sources and identify of the restriction fragment that contains the sequence.
What method is used to identify the presence or absence of a specific nucleotide sequence of DNA?
Southern blotting
What method is used to identify the size of a restriction fragment that contains a desired gene?
Southern blotting
Outline the southern blotting process.
DNA isolated from each source.
DNA digested by specific restriction enzyme.
Restriction fragments loaded onto electrophoresis gel.
Fragments of different base length separated by electrophoresis.
DNA transferred onto nylon filter.
Radioactively labelled nucleic acid probe added and binds to complimentary DNA sequences.
Nylon filter covered with X-ray film to identify the location of the probes and therefore the desired DNA fragments.
Shows which sources of DNA contain the desired DNA fragment.
What is used to identify the presence of a specific gene sequence in southern blotting process?
Nucleic acid probes which can be seen with an X-ray film.
What dye is used to made the DNA fragments visible in southern blotting?
Ethidium bromide
What is the purpose of Ethidium Bromide in southern blotting?
Makes the DNA fragments become visible.
What enzyme types are used to digest DNA in southern blotting?
Restriction enzymes
What type of filter are DNA fragments loaded onto in southern blotting?
Nylon filter
Why are agar plates incubated for 16-24 hours during blue white screening?
Gives enough time for bacteria to form colonies and show as coloured if appropriate.
Which genome is the lac operon naturally found in?
E.coli
Which section of the lac operon encodes for beta galactoside?
LacZ
What is the purpose of the LacI section of the Lac operon?
Codes for repressor proteins
What is the purpose of the promoter region of the lac operon?
Binding site for transcription enzyme and RNA polymerase.
What is the purpose of the operator region of the lac operon?
Binding site for lac repressor enzyme.
What is the purpose of the Lac Z region of the lac operon?
Encodes for beta galactoside enzyme.
What are the main components of the lac operon?
LacI, Promoter region, operator region, LacZ
The active form of beta galactoside contains 4 identical subunits. What is the collective name for these subunits?
Homotetrameric
How many subunits does the active form of beta galactoside have and describe the similarity of the subunits.
4 identical subunits
What type of enzyme is beta galactoside?
Homotetrameric
What is the overall function of the beta galactoside enzyme?
Breaks glycosidic molecules within substrate molecules.
Give an example of a substrate which beta galactoside hydrolyses
X-Gal
When does X-gal change from colourless to dark blue?
When its glycosidic bonds are hydrolysed by beta galactoside.
Where does the lacI repressor protein bind on the lac operon?
Operator sequence
What is used to remove LacI repressor proteins from the lac operon?
IPTG reagent
How is beta galactoside concentration varied due to the presence of IPTG at the lac operon.
When IPTG is added, the repressor proteins is removed from the lac operon. This means more DNA polymerase can bind and increase transcription of Lac Z to beta galactoside.
Activates beta galactoside production.
Describe a structure needed by the E.coli used in blue white screening and what does this do?
LacZ deletion mutation
Codes for beta galactoside enzymes which are non-functional and can’t form a homotetrameric structure.
How can the functionality of beta galactoside enzymes be re-activated if the lac operon has a LacZ deletion mutation?
Adding a short, specific, alpha peptide to undergo alpha complementation.
What is alpha complementation in blue white screening?
The addition of a short, specific, alpha peptide to allow beta galactoside to become active.
When a specific DNA segment is being inserted into a plasmid, why is the genetic information incubated at a low heat such as 16 degreed Celsius?
This allows hydrogen bonds to form complementary base pairs between the sticky ends of the plasmid and the DNA.
What is ligation?
The forming of permanent covalent bonds between two fragments of DNA using DNA ligase.
What type of bonds does DNA ligase catalyse the formation of?
Covalent phosphodiester bonds
Why does DNA ligase need a cofactor to work and what does it do?
Cofactor increases the efficiency of DNA ligase by providing free energy in the form of a high energy phosphate linkage.
What specific cofactor does DNA ligase use?
ATP
When is relegation needed in the formation of rDNA?
When the plasmid and DNA have been cut with blunt ends.
What is the disadvantage of using relegation when forming rDNA?
It reduces the proportion of recombinant plasmids and therefore reduces cloning efficiency.
How can relegation be prevented in the process of making rDNA?
Remove the 5’ phosphate group from the plasmid before ligation using phosphatase enzyme.
How does removing a phosphate from the 5’ group of a plasma prevent relegation when forming rDNA?
DNA ligase cannot catalyse formation of a phosphodiester bond so relegation cannot occur.
What would happen if both the plasmid and DNA insert were treated with phosphatase before ligation when forming rDNA?
No phosphodiester bonds would form therefore ligation couldn’t occur and no rDNA would be formed. No DNA cloning.
What is used to spread bacteria out on an agar plate?
L spread
How many bacterial colonies will form in bacterial cells with no plasmids taken up ? (formation of rDNA)?
None
Why must a buffer be present during gel electrophoresis?
If the solution becomes too acidic or alkaline, the DNA may be denatured in terms of its structure or charge.
Describe the location of the positive and negative electrodes on a gel electrophoresis set-up.
Negative electrode - side with wells (top)
Positive electrode - side without wells (bottom)
What causes DNA to move through a gel electrophoresis set-up?
DNA has a negative charge (on its phosphate backbone) at the pH of the gel electrophoresis set-up and is therefore attracted and migrates towards the positive electrode.
What Determines the distance that a DNA sequence moves on a gel electrophoresis set-up?
It’s length.
Shorter DNA sequences move further in a given time than longer DNA sequences.
What is meant by ethidium bromide being an intercalating agent?
Capable of inserting itself between the bases of DNA.
What is needed to visualise DNA fragments dyed with ethidium bromide?
UV light
Why is Blue White Plating described as a ‘shotgun’ DNA analysis approach?
It produces a picture of random DNA from the whole genome, not select fragments.
What are bacteriophages?
Viruses that infect bacterial cells.
Why are bacteriophages often used instead of bacteria as vectors?
More efficient because they use transduction rather than transformation.
Can replicate larger fragments of DNA.
Where is rDNA found in bacteriophages?
In their heads.
What method is used to screen for the desired genes in bacteriophage plaques?
Hybridisation
What issues do restriction enzymes have when forming rDNA?
They do not respect gene boundaries so some genes in the gene library may be divided up among 2 or more clones and therefore be in different plasmids of bacteriophage heads.
Why is cDNA often used over rDNA?
Functional proteins won’t be made by rDNA if the introns are present in the DNA because bacteria can’t remove introns.
What is the benefit of having a large genetic insert when forming a genome library?
Minimises the number of clones required to make up the genomic library.
Why is it an issue to use large DNA molecules in gel electrophoresis?
Too many bands will form and will appear as a smear - no distinct DNA ladder.
What is the starting molecule of retro-grade DNA replication?
mRNA
What is required for retro-grade DNA replication?
A primer
Reverse transcriptase
What is the product of retro-grade gene isolation?
cDNA
What is the overall purpose of reverse transcriptase?
Synthesises DNA from mRNA.
Outline the process of retro-grade to produce cDNA from mRNA.
1) Reverse transcriptase is added to a test tube containing isolated mRNA from a cell sample.
2) Reverse transcriptase makes the first DNA strand using the mRNA as a template and a short pay-dT as a DNA primer.
3) mRNA degraded by another enzyme.
4) DNA polymerase synthesises the second DNA strand using a primer in the reaction mixture.
5) cDNA produced
What is cDNA?
A DNA copy of mRNA produced by reverse transcriptase and DNA polymerase.
What modifications must be made to cDNA before it can be inserted into a vector?
Restriction sites added to each end.
What are cDNA libraries used for?
Studying specialised functions of specific cell types.
Used when only interested in the coding sequences of DNA.
Studying gene expression at different stages of development in an organism.
Do cDNA genome libraries contain non-coding regions?
No
When are rDNA gene libraries used?
If not much is known about the gene of interest.
If you are interested in looking at non-coding regions of the DNA.
Do rDNA genomic libraries contain non-coding regions?
Yes
What is an Oligonucleotide?
Single stranded molecule with a relatively small number of nucleotides.
How are nucleic acid probes traced during nucleic acid hybridisation?
The probes will complimentary base pair to the single stranded DNA of the gene of interest. They are then traced by radioactive labelling and a fluorescent marker.
Outline the process of Nucleic acid hybridisation.
1) Denaturation to separate double stranded DNA into single strands using chemicals or hear.
2) Probes are incubated on filter paper.
3) Probe complimentary base pairs with complementary DNA on filter.
4) Excess DNA rinsed off
5) Filter laid on film to detect radioactivity - autoradiography.
What method is used to identify the nucleic acid probes during nucleic acid hybridisation?
Autoradiography - probes are radioactive and can be seen using X-ray film.
What is autoradiography?
Using X-ray film to visualise molecules that have been radioactively labelled.
Outline the process of colony hybridisation.
Bacterial colonies containing cloned segments of foreign DNA are transferred to a filter. The cells are then treated to denature double stranded DNA to become single stranded DNA.
Nucleic acid probe is added.
Autoradiography used to identify presence of desired gene.
Why is DNA denaturation required in hybridisation of DNA?
Separating the strands of DNA allows a nucleic acids probe to bind.
What techniques can be used to analyse the DNA in specific genes?
Gel electrophoresis
Restriction Enzymes
Southern Blotting
What does the migration of DNA samples depend on during gel electrophoresis?
Length of DNA molecule (number of bases), charge
In gel electrophoresis, what prevents the longer fragments moving through the gel at a fast pace?
Mesh of polymer fibres
How are the different bands of gel electrophoresis identified?
The bands become fluorescent under UV light.
What is PAGE used to identify?
Small molecules of DNA or RNA.
What is restriction fragment analysis used for?
Used to detect differences in nucleotide sequences of DNA molecules.
Outline the process of restoration fragment analysis.
1) Treat 2 DNA samples with the same restriction enzyme.
2) Slightly different alleles will have differences in their restriction sites.
3) Produce different band patterns in gel electrophoresis.
4) Electrophoresis used to identify differences in the restriction sites.
What are the negatives of using restriction fragment analysis and gel electrophoresis alone?
They will produce too many DNA fragments to identify desired individual genes.
What 2 methods are combined to form the ‘Southern blotting’ method?
Gel electrophoresis and Nucleic acid hybridisation
When was the Southern Comfort method of DNA analysis produced?
1975
What can the ‘Southern blotting’ method identify?
Presence of specific DNA fragments in a sample, number of specific DNA fragments, size of restriction fragments that contain specific DNA fragments.
Outline the process of using bacteriophages as vectors for DNA cloning.
1) The phage binds to the surface of the bacterial cell and injects it’s DNA.
2) Host DNA digested
3) New phage DNA forms using nucleotides from the formed host DNA.
4) Host cell transcribes and translates the phage DNA producing phage proteins.
5) Assembly of a new phage is complete. A phage-enclosed enzyme causes the bacterial host cell to lyse.
Outline the process of ‘Southern Blotting’.
1) DNA is isolated from each source and digested using specific restriction enzymes.
2) The produced restriction fragments are laded onto a gel electrophoresis plate with an alkaline buffer.
3) Fragments are separated by gel electrophoresis radioactively labelled nucleic acid probes (ethidium bromide) are added.
4) Transfered to nylon filter
5) X-ray film used to identify the location of the desired genes - Autoradiography.
What is the purpose of a buffer in gel electrophoresis?
Creates a ion source which allows charge to flow through the plate.
Ensures DNA is not denatured - maintains pH.
What are RFLPs?
Restriction fragment length polymorphisms - differences in restriction fragment patterns throughout a genome.
What are the advantages of polymorphisms for the southern blotting process?
Different alleles can serve as genetic markers for particular locus of the genome.
Do southern blotting methods contain non-coding sections of DNA?
Yes
What is a DNA microarray slide?
A small cilia or glass slide containing thousands of spots.
What type of DNA is contained within each spot of a microarray?
Single stranded, synthetic DNA with a known sequence. These fragments are from many different genes.
What is expression of genes dependent on?
Cell type and environmental conditions.
Outline how the DNA used in microarray analysis methods is synthesised.
mRNA is isolated from particular cell types or cells that have been subjected to an environmental agent.
Fluorescently marked DNA nucleotides complimentary base pair with the RNA to form cDNA. (This is done in a test tube).
The cDNA hybridises with the single-stranded DNA in the microarray spots by complimentary base pairing.
How can the hybridised DNA samples of microarray be seen?
They can be seen under a UV light because the cDNA will act as a probe with fluorescent markers and glow green.
What must happen before a microarray slide is viewed under a UV microscope and why?
Washing away of excess cDNA so that areas of the microarray plate only glow when base pairing between the cDNA and DNA of known fragments have bonded.
What is the microarray technique used to study?
Many genes from a particular cell at the same time.
Give some applications of PCR.
DNA cloning
For medical diagnosis
Used in forensic DNA analysis
Outline the process of PCR.
- Denaturation - Double stranded DNA is separated at 96 degrees Celsius.
2 single strands of DNA are produced. - Primer annealing at 55-65 degrees Celsius.
Short, specifically chosen DNA primers bing to the gene fragments of interest by complimentary base pairing. - Primer extension at 72 degrees Celsius.
The tax polymerase enzyme adds DNA nucleotides. - 2 Double strands of DNA are produced.
- Cycle repeated
What temperature is DNA denatured at during PCR?
96 degrees celcius
What temperature does Primer annealing occur at during PCR?
55-65 degrees celcius
What temperature does Primer extension occur at during PCR?
72 degrees Celsius
What DNA polymerase enzyme is used for PCR and why?
DNA Taq polymerase
It is heat resistant
At what temperature is Taq polymerase most active?
About 70 degrees Celsius.
Who devised the PCR method and in what year?
Kary Mullis
1983
Where does the PCR reaction take place?
In vitro - in a test tube
Why is a thermocycler used in PCR?
It heats and cools the reaction test tubes in a short amount of time.
What is the benefit of using PCR over recombinant plasmids?
Every colony produced by the bacteria should contain plasmids with the desired genes - no plasmids without the desired gene. Therefore alot more efficient.
What components are required to carry out PCR?
DNA source.
Supply of nucleotides.
Heat resistant DNA polymerase (Taq polymerase).
DNA primers.
What is the purpose of DNA primers in PCR?
They initiate DNA synthesis of the chosen DNA fragment by adding complimentary sequences to the 3’ end of the target region.
How does DNA (Taq) polymerase undergo annealing in PCR?
Adds nucleotides to the 3’ end of the strand therefore copying the target sequence.
Why is the DNA denatured in PCR?
Makes the DNA become accessible by primers.
Which end of the DNA molecule does Taq polymerase add nucleotides to in PCR?
3’
What is meant by PCR causing an exponential increase in DNA?
Increase quickly by large amounts.
Why does PCR not replace gene cloning?
Mistakes are often made during PCR meaning that PCR is just used to provide more specific DNA fragments that can be used to clone genes.
What are the advantages of PCR.
Highly specific.
Only a tiny sample of DNA is required.
What are some uses of PCR?
Used to diagnose prenatal genetic disorders by sampling DNA from embryonic cells.
Used to detect virally infected cells.
Amplifying fragments of DNA which are used to study evolution.
Forensic analysis - amplifies small samples of DNA from blood, hair or semen.
DNA fingerprinting - to make DNA profiles.
What cells are DNA samples from to diagnose prenatal genetic disorders by PCR?
Embryonic cells
What is the overall main purpose of DNA microarray assays?
To compare patterns of gene expression in different tissues at afferent times or under different conditions.
What are DNA microarray assays used to identify or produce?
Used to discover how genes interact to form complete living organisms.
Studying the differences in gene expression.
Could lead to new diagnostic methods or therapies.
Outline the process of carrying out a DNA microarray assay.
Notes
What is a DNA chip in a microarray assay?
The single stranded DNA molecules representing all the different genes, fixed onto a glass slide and tightly packed onto an assay grid.
What does each spot on a DNA chip represent?
A gene
What is the purpose of RNA sequencing?
Sequencing cDNA samples fro different tissues or embryonic stages to determine the gene expression differences.
Outline the process of RNA sequencing.
- mRNAs are isolated from the tissue being studied.
- mRNAs are cut into similar-sized, smaller fragments.
- mRNAs are reverse transcribed into cDNAs of the same size.
- cDNAs are sequenced.
- The short sequences are mapped by a computer onto the genome sequence.
What do black areas on a DNA microarray show?
Areas where neither the control nor sample DNA is expressed.
What does the location and colour of each array in a microarray show?
Tell us whether a gene or mutation is present in the control or sample DNA.
The colours represent either healthy(control) or diseased (sample) tissue.
What is the purpose of in vitro mutagenesis?
Determining gene functions by disabling genes and observing their consequences.
In general, how does ‘In vitro mutanagenesis’ work?
Specific mutations are introduced into a cloned gene. They then either alter or destroy the function of that gene. When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype.
What is the CRISPR-Cas 9 system ?
A technique for editing genes in living cells and organisms.
What is gene therapy?
A treatment where a patient’s affected cells’ genotype is altered by the addition or deletion of a specific gene.
What is a therapeutic gene?
A normal version of a gene carried within a genetically engineered virus. This is used to then infect malignant cells.
What are the issues with gene therapy?
Problems with placement of genes and control of gene expression.
Ethical issues.
What conditions must be met before gene therapy can be carried out on an individual?
Disease must be serious and incurable OR have chronic conditions requiring lifelong treatments.
The gene causing the disease must have been identified and understaff.
What is the cause of SCID?
Children with SCID lack a key enzyme involved in maturation of cells from the immune system.
Outline how Ex-vivo gene therapy is used with the p53 gene.
Using cells from a tumour with the mutant tumour-suppressor p53 genes. The cells are grown in large numbers in vitro. The cells are then incubated with retroviruses containing normal versions of the p53 gene.
The transformed cells are then returned to the patient, hopefully returning expression of tumour-supressor protein activity.
What are the negatives of Ex-vivo gene therapy ?
Only limited success so far.
What is the purpose of using Ex-vivo gene therapy on the p53 gene?
Causes mutated tumour suppressor genes to become functional and therefore allow tumour-supressor proteins to function.
In each cycle of PCR, how should the quantity of DNA change?
It should double
What are amplicons in PCR?
Copies of DNA that have been produced.
What is the most common method of analysis of amplicons of PCR?
Gel electrophoresis
How many primers are required for PCR to work and where on the DNA sequence do they both bind?
2 - they bind to the DNA at the start and end of the sequence which needs to be amplified.
Where does Taq polymerase derive from ?
A species of bacteria (Thermus Aquaticus) that live in hot springs.
What is the normal denaturing temperature of a protein?
Approximately 65 degrees
After how many cycles can you begin to produce the desired gene copies in PCR?
3
What is the purpose of destining PCR primers that include restriction sites?
This allows the products of PCR to be cloned into plasmic vectors for further gene cloning.
Each colony produced should contain recombinant plasmids with the desired genetic sequence. Highly efficient.
If you are studying more than one cell type via microarray methods at once, what modifications must be made?
You must add a different fluorescent dye to each of the cells.
You must add equal volumes of each DNA sample.
If there is very bright spot on a microarray after hybridisation, what does this mean?
There is high expression of that particular gene, perhaps over-expression.
Why is the Cas9 protein often used in DNA analysis?
It has an active site which can cleave (cut) DNA.
(Similar to the function of a restriction enzyme).
What type of cells does the CRISPR-Cas9 analysed the DNA of?
Living cells
How are children with SCID treated?
If bone marrow is removed from a patient, an RNA retrovirus can be used to insert the normal, functional, un-mutated gene into the bone marrow cells.
This means the cells of the immune system are able to mature properly again.
What is meant by primers ‘flanking’ the DNA in PCR?
One primer anneals to either side of the gene section you wish to amplify.
In what year were insulin injections first administered and what type of insulin was used?
1922 - pig insulin
How does pig insulin differ from human insulin?
Differs by one amino acid. (Bovine by 3 changes to ovine by 4).
How does injecting pig insulin into a patient aid their immunogenic response?
When pig insulin is injected several times a day, it promotes a strong antibody response.
Describe the relative immunogenic strengths of human and pig insulin.
Pig insulin is stronger than human insulin.
