Topic 8 B: Genome Projects and Gene Technologies Flashcards
Cause of Type 1 diabetes?
- lack of insulin production
- it is released in response to high blood glucose levels in order to reverse this change
How is type 1 diabetes treated?
- insulin injections
- previous treatments involved extracting protein from human / animal donors and introducing it to the patient. However, can cause rejections or infections
Recombinant DNA definition?
Dna of two different organisms that have been combined
What is recombinant dna technology?
involves transferring a fragment of dna from one organism to another
Transgenic / genetically modified organism definition?
The resulting organisms produced as a result of technology.
How is DNA of one organism accepted by a different species?
- genetic code is universal - same in all living organisms
- resulting protein produced is also functional as transcription and translation is the same in all living organisms
Process of making proteins using recombinant dna technology?
- isolation - of the dna fragments that have the gene for desired protein
- insertion - of dna fragment into vector
- transformation, - the transfer of dna into suitable host cell
- identification - of host cells that have successfully taken up gene by using gene markers
- growth / cloning - of population of host cells
Methods of identifying and isolating gene that codes for protein?
- conversion of mRNA into cDNA using reverse transcriptase
- using restriction endonucleases to cut fragments containing the desire gene from DNA
- creating the gene in a gene machine, usually based on known protein structure
What is reverse transcriptase and its overall function?
- enzyme found in some viruses e.g., HIV (retrovirus)
- ## catalyse the reverse of transcription (RNA into DNA)
How does reverse transcriptase produce specific dna fragment?
used to produce a dna strand from mRNA synthesised in the cell.
dna polymerase then produces double stranded cDNA.
1. mrna acts as template (from insulin)
2. complementary copy of cdna formed by enzyme and binds
3. single stranded cdna is isolated by hydrolysis of mrna with an enzyme
4. double stranded dna is formed on template of cdna using dna polymerase
5. result = copy of human insulin gene
Advantages and disadvantages of using reverse transcriptase?
adv = mrna is present in cells from genes that are being transcribed actively so an easy and plentiful source of desired mrna
disadv = time consuming - multiple steps
What are restriction enzymes and what are they used for?
- bacteria produce these to protect themselves from invading viruses called bacteriophages
- used by bacteria to cut up viral dna
- they cut as specific (active site complementary to specific bases), palindromic (sequence reads same in antiparallel directions) sites
Restriction enzymes and cleaving?
- blunt ends - cut straight through both chains
- sticky ends - staggered cut in two chains creating single-stranded overhangs of DNA
Significance of sticky ends?
- have a strand of single stranded dna which are complementary to each other
- they will join with another sticky end cut with the SAME restriction enzyme
Advantages and disadvantages of restriction enzymes?
adv = if dna and vector cut with the same restriction enzymes, sticky ends can make it easier for dna to be inserted into vector
disadv = fragments produced still contain introns
Gene machine process?
- amino acid sequence of desired protein determined
- from this, mRNA codons that code for each amino acid are looked up
- complementary dna triplets then worked out
- dna sequence is fed into computer which generates series of small overlapping nucleotides (oligonucleotides), which can be used to produce desired genes
- oligonucleotides joined together to make the gene, this gene contains no introns
- gene is replicated using PCR technique
- using sticky ends, the gene is inserted into a plasmid for cloning
Advantages and disadvantages of gene machine?
adv = any dna fragment can be produced and modified.
adv = short time needed
adv = very accurate
adv = free from introns and non-coding regions so can be transcribed and translated by prokaryotic cells too
disadv = need to know exactly which bases / codons code for desired amino acids
in vivo and in vitro cloning?
in vivo = transferring the DNA fragments to a host cell using a vector (inside)
in vitro = using the polymerase chain reaction (inside living cells)
What is a vector?
Used to transport DNA into a host cell.
What is a plasmid?
Most commonly used vector.
Why are Plasmids useful as vectors?
- big enough to hold genes
- often they are closed loops of DNA which are less likely to be broken down
- they contain control sequences such as transcription promoter sequences
- have marker genes, so you can tell which of the host cells have taken up the vector
How is DNA inserted into a vector?
- Desired gene is isolated using restriction enzymes. This produces a DNA fragment with sticky end.
- Plasmid (containing gene for antibiotic resistance A - tetracycline, B - ampicilin) is cut with the same restriction enzymes. This produces complementary sticky ends.
- the DNA from both sources attach together WITH ONLY TETRA, temporarily, until more permanent covalent bonds can be formed between the sugars and phosphates
- the role of DNA ligase is to rejoin the sugar phosphate backbones by formation of phosphodiester bonds betweenn dna fragment and tetra
- Recombinant tetra
What is the promoter and terminator region?
P = binding site for RNA polymerase, so that transcription can take place
T = releases RNA polymerase to end transcription
How is recombinant DNA introduced into the host?
- transformation
- chemical or electroporation
- in chemical transformation = ice-cold calcium ions soak the bacterial cells which increases the permeability of the membranes, allowing the recombinant DNA to enter the host bacterial cells. A heat shock is often used to encourage uptake.
- electroporation promotes transformation through an electric shock to create holes in cell membrane to allow recombinant DNA to enter
Why wont all bacterial cells take up the plasmids?
- some plasmids closed up again without incorporating the DNA fragment
- sometimes the DNA fragments ends jons together to form its own plasmid
What happens when DNA fragments are added to plasmind with antibiotic resistance genes?
- it interrupts one of the antibiotic resistance genes (Tetracycline), but the other remains unaffected (Ampicilin)
1. bacteria are grown on a medium that contains the antibiotic ampicilin
2. bacteria that have taken up the plasmid will survive as they contain the antibiotic resistance genes
3. the bacteria that have not taken up the plasmid will die
The recombinant plasmid is reintroduced into bacteria cells, which are the three types?
- bacteria without plasmids - it will be killed if exposed to either ampicilin or tetracycline
- bacteria that has taken up plasmid but not recombinant one - resistant to both so will survive if exposed to either
- bacteria that has taken up recombinant / transformed plasmid - resistant to only ampicilin, it will be killed if exposed to tetracycline
How to find out what bacteria cells have taken up the correct plasmid and types?
Use a second gene as a marker
- 2nd antibiotic resistance gene
- gene coding for a fluorescent protein
- gene that produces an enzyme with an identifiable action
How does antibiotic resistance marker - replica plating work to find correct plasmid?
- spread all bacterial cells over agar containing ampicilin
- bacteria colonies that grow will be ones that took up plasmid, ones that didnt will die. however, dont know if these bacteria contain a hybrid (recombinant) plasmid or a normal one
- sterilized velvet surface stuck to original bacteria agar plate and moved placed onto replica plate of agar containing tetracycline
- compare the original plate (Amp) and replica plate (Tetra) and look for bacteria that grew on Amp but not Tetra as that bacteria has the inserted gene in it so the tetracycline has been destroyed
- identify this bacteria and then take sample of this correct plasmid bacteria and grow it in bulk
How do enzyme markers work to find correct plasmid?
- some plasmids contain gene that codes for lactase enzyme - turns substrates from colourless to blue
- gene you want to clone is inserted into the plasmid, into the lactase gene (using same restriction enzymes to make complementary sticky ends, and DNA ligase to rejoin nucleotides)
- bacterial colonies are now grown on agar containing colour-changing substrate
- colonies where substrate turned blue do not have correct recombinant plasmid, vice versa
How do fluorescent markers work to find correct plasmid?
- desired gene is inserted into site of green fluorescent gene (GFP)
- disrupts the expression of this gene
- when UV shone upon bacterial colonies, the correct plasmids will not fluoresce
Exam question: Molecular biologists often use plasmids which contain antibiotic resistance genes. Explain why? (2)
- act as marker gene
- allows detection of cells containing plasmid
Exam question: Give one advantage of using fluorescent gene markers rather than antibiotic resistance gene markers?
results can be obtained more easily and more quickly - because with antiobiotic resistance markers, the bacterial cells with desired gene are killed, so replica plating is necessary to obtain cells with the gene. With fluorescent markers, bacterial cells arent killed so no need to carry out replica plating
What is polymerase chain reactions (PCR)?
laboratory procedure through which small samples of DNA can be cloned, or amplified to make many copies.
PCR and significance of number of DNA produced?
each cycle doubles the number of DNA molecules
2 to the power of N (number of cycles) to find how many DNA molecules produced all together
e.g., 6 cycles = 2^6 = 64
What substances need to be mixed together for PCR to occur?
- DNA that needs to be amplified
- free nucleotides
- dna polymerase
- primers - short, single-stranded DNA molecules that act as starting point to make new DNA molecules
- suitable buffer solution at optimum enzyme pH
Stage one of PCR?
Separation of the DNA
- heat to 95C
- breaks hydrogen bonds holding complementary bases together so makes DNA single-stranded
Stage two of PCR?
Annealing of the primers
- mixture cooled to 55C
- primers are able to anneal (join) to comp bases at ends of DNA fragment - joined by hydrogen bonds
- DNA polymerase can only add nucleotides to ones already there
-
Stage three of PCR?
Extension / synthesis of DNA
- increased temperature to 72C
- Taq DNA polymerase adds nucleotides to primers by creating phosphodiester bonds (condensation reaction) (quick process)
How can Stage three of PCR happen at such high temperatures without denaturing enzymes?
- DNA polymerase used is from bacteria Thermus aquaticus - lives in hot springs so enzymes are heat-stable
- Taq DNA polymerase has high optimum temperature, so heat to 72C otherwise activity too sloww
Why is PCR called a chain reaction?
number of molecules increases exponentially
Exam question: What is the role of primer and DNA polymerase in PCR process?
primer = enables replication / sequencing to start
DNA polymerase = joins dna nucleotides
Exam question: suggest one use of PCR?
replication of DNA from crime scenes / tissue sample / for dna sequencing / gene cloning
Exam question: Give two ways in which PCR is different from transcription?
- T uses RNA polymerase, not DNA polymerase
- T uses RNA nucleotides / uracil, not DNA nucleotides / thymine
- T uses one template strand, PCR uses both
T uses enzymes to seperate strands, PCR uses heat
Exam question: Describe how the PCR is carried out?
- DNA heated to 95C
- strands seperate
- cooled to 55/65C
- primers bind
- nucleotides attach
- by comp base pairing
- temperature rise to 72C
- Taq DNA polymerase joins nucleotides
- cycle repeated
Advantages and Disadvantages of In vitro gene cloning?
adv = rapid - 100 billion copies made in hour
adv = useful for analysing very small quantities of DNA e.g., samples from crime scenes
adv = doesnt require living cells - no complex culturing techniques that require time / effort
disadv = PCR will also amplify any other contaminating DNA found at scene (requires pure sample)
Advantages and Disadvantages of in vivo gene cloning?
adv = can be used to introduce a gene into another organism
adv = almost no risk of contamination - restriction endonucleases / enzymes can match sticky ends of plasmid and DNa so any contaminant of DNA will not be taken up by plasmid
adv = accurate
adv =precises - produces copies of specifically cut out gene rather than copies of DNA samples
adv = produces transformed bacteria that can be used to produce large quantities of gene products for medical / commercial use
disadv = takes more time than PCR to amplify DNA
disadv = requires living cells / organisms
Exam question: explain why two different primers are used in PCR?
because the sequences at the opposite ends of the two strands of DNA are different
What does genetic origin mean?
Genetic diseases are caused by gene mutation
What is recombinant DNA technology used for in relation to genetic disorders?
diagnosing and treating genetic disorders
- must find out where the mutation has taken place by using DNA probes
What is a DNA probe?
- short, single-stranded length of DNA
- its DNA is complementary to section of DNA that is being searched for
- produced in small quantities but are amplified by PCR
How are DNA probes labelled?
- radioactive - 32P isotope incorporated into nucleotides which can then be viewed using an autoradiograph
- fluorescent dye - glows green when certain wavelengths of light strike it
How is DNA hybridisation used to locate specific alleles?
- sequence of nucleotides on mutated gene is determined through dna sequencing
- fragment of dna with comp bases to mutant allele of gene is produced
- dna probe is formed by fluorescently labelling dna fragment
- pcr technique used to produce multiple copies of dna probe
- probe is added to single-stranded dna fragment from person being screened
- if donor has mutated gene, some donor dna fragments will have comp base sequence to the probe and they will bind / hybridisation
- these dna fragments now labelled with fluorescent probe and can be distinguished
What is genetic screening?
using dna probes to analyse peoples dna
What is genetic screening used for?
- genetic disorders - screens potential carriers to determine if parent is carrier and if child may be affected by genetic disorder
- cancer - screen for mutated oncogenes and tumour suppressor genes (in order to develop tumour, person needs to inherit mutations in both alleles for these genes. carrying one mutant allele puts these individuals at greater risk of developing cancer)
- personalised medicine - screen to provide advice and determine most effective dose of a drug or effects of certain drugs on the body (adv = save time and money and drugs)
What comes after genetic screening and describe it?
Genetic counselling
- if people find out they are carriers of a genetic disorder, higher risk of cancer or not responsive to certain drugs, they can be given medical advice on next steps
- e.g., abortion, surrogacy, IVF, most effective form of treatment, risk factors for developing cancer (smoking) etc
Exam question: How might a person who has be screened and found one mutant allele of tumour suppressor gene act?
- might change lifestyle to reduce risk of cancer e.g., stop smoking, lose weight, eat healthier, avoid mutagens
What is genetic fingerprinting used for?
diagnostic tool used in forensic science, plant and animal breeding, and medical diagnosis
What does genetic fingerprinting rely on?
dna of every individual except identical twins is unique
What dna sequences does genetic fingerprinting use?
- repetitive, non-coding sequences of dna called variable number tandem repeats (VNTRs)
- every human has unique pattern for the number and lenght of VNTRs
- more closely related individuals are, more similiar the VNTRs are
Steps involved in genetic fingerprinting?
- extraction
- digestion
- separation
- hybridisation
- development
How do we produce genetic fingerprinting?
- Extraction - DNA is extracted from sample
- Digestion - restriction endonucleases cut dna into fragments
- Separation - fragments are separated using gel electrophoresis, then immersed in alkali to seperated double strands into single
- DNA fragments transferred from gel to nylon membrane
- Hybridisation - DNA probes are added to label the fragments, these radioactive probes attach to specific fragments
- Development - membrane with radioactively labelled DNA fragments is placed onto an x-ray film. development of x-ray film reveals dark bands where the radioactive DNA probes have attached
Exam question: explain why radioactive DNA probes are used to locate specific DNA fragments?
- DNA invisible on gel
- allows detection
Exam question: Explain why it is often necessary to use the pcr when producing genetic fingerprint?
PCR used to increase quantity of DNA because quantity available is often small e.g., crime scenes
How does gel electrophoresis work?
- electric field used to move / separate molecules
- purpose = separate pieces of dna in order to visualise them and determine their size
- dna moves to the positive electrode because it is negatively charged
- size of the dna molecules affects speed of dna through gel, small molecules will travel faster and further than long ones
Exam question: Explain how electrophoresis separates the fragments of DNA?
- move towards positive electrodes as it is negatively charged
- speed is determined by size of dna molecules - smaller = faster
How to interpret results of genetic fingerprinting?
- dna fingerprints of two samples are visually compared
- if there is a match in patterns, each fingerprint is passed through a scanning machine which calculates length of DNA fragments
- the odds of someone else having same fingerprint is calculated - the closer the match between the pattern the greater the probability that they came from the same person
How is genetic fingerprinting used in genetic relationships and variabilty?
- individuals inherit half of genetic material from each parent, therefore bands on gel should similiar to parents fingerprints - used to determine parentage
- can also be used as a measure of variability, a population with low genetic diversity would have similar genetic fingerprints vice versa
How is genetic fingerprinting used in forensic science?
- dna left at crime scene used to generate a fingerprint, then dna fingerprints used from different suspects and compared to crime scene dna
- close match doesnt prove guilt, dna may be present as they are a relative or dna may have been contaminated
How is genetic fingerprinting used in medical diagnosis?
- can be used to identify microbial inefction if patients microbes fingerprints are matched to fingerprints of known pathogens
How is genetic fingerprints used in plant / animal breeding?
- used to identify if plants or animals have particular allele of a desirable gene, these individuals can be selected for breeding to increase frequency of desirable allele
- can also be used to determine the paternity of animal for pedigree
