8.4 gene technologies Flashcards
What is recombinant DNA technology?
Extracting and transferring genes from a donor org to a recipitant org
What does the recipiant org become after recombinant DNA technology?
a genetically modified org
Why is recombinant DNA technology possible? (2 reasons)
- the genetic code is universal to all orgs
- transcription and translation mechanisms are mostly the same in all orgs
What are the 2 area of uses of recombinant DNA technology?
agriculture and medicine
What is an example of using recombinant DNA tech in medicine?
treatments for Type 1 diabetes:
- the insulin in the injections if from prokaryotes that have had human DNA transfered into them so that they can produce human insulin
What is an example of the use of recombinant DNA tech in agriculture?
genetic modification of animals and plants to acquire new characteristics
eg. disease-resistant crops and nutritional crops
What is donor DNA?
gene that is isolated for insertion
What is a vector?
a carrier:
plasmids carry DNA into the recipiant DNA
What is restriction endonucleases?
enzymes that cut DNA at specific restriction sites
What are DNA ligases?
enzymes that join sections of DNA together
What are sticky ends?
2 ends of ‘cut’ DNA segment that have unpaired bases
What is recombinant DNA?
DNA which is formed when a piece of foreign DNA is incorporated into the plasmid from a bacterium
What is reverse transcriptase?
enzymes used to synthesise DNA from mRNA in specific cells
What is a clone?
a population of genetically identical cells or orgs
What are the 5-step processes of recombinant DNA tech?
- isolation
- insertion
- transformation
- identification
- culturing
What are the 3 different methods that are used to isolate desried genes?
restriction endonucleases
reverse transcriptase
gene machine
What 2 things need to be done before you isolate a desired gene?
- locate the gene - use a cell that naturally produced the desired protein
- identify the gene locus by using a gene probe
How does a gene probe identify a gene locus?
the probe has a specific seq of single-stranded DNA which is complimentary to the desired gene
it is labelled by using flouresence or a radioactive tracer
What type of org naturally produces restriction endonucleases and why?
bacteria as a defence mech against viral infection
How do restriction endonucleases cut double stranded DNA?
hydrolyse phosphodiester bonds
What are the 2 different ways that restriction endonucleases cut DNA ( the structures DNA forms after being cut)?
form blunt ends
or palindromic sticky ends = staggered cut
What does palindromic mean?
the DNA base sequences of the staggered sticky ends read the same forwards and backwards
Which type of restriction endonuclease cutting is more useful?
staggered sticky ends
Why are staggered sticky ends more useful?
they have unpaired DNA bases that can easily complimentary base pair to exposed bases on DNA in the recipiant org
easier to join DNA
Where is RNA reverse transcriptase found?
in retroviruses for replication in a host cell
How is reverse transcriptase used to isolate the desired gene?
- there are 2 copies of the gene on a DNA helix - one on each strand
- mRNA is transcribed from one DNA template strand
- reverse transcriptase runs antiparallel to mRNA to form a single strand of cDNA by complimentary base pairing w/ the mRNA
- DNA polymerase is added to form dsDNA
- ds cDNA can be inserted into a vector
What are the advantages of using reverse transcriptase to isolate the gene?
- there is more RNA carrying the desired gene
- RNA w/ desired gene is found in the cytoplasm = easier to isolate the gene from rather than going through the nucleus to get the DNA
- mRNA has had its introns removed so there will be no introns in the bacteria as they wouldn’t know what to do w/ introns
How is a gene machine used to isolate a gene?
- find the primary structure of the desired protein, then the a.a seq, the mRNA codon seq to find the DNA triplets coding for the desired gene producing that protein
- feed the DNA triplets into a computer
- short fragmnts of the desired gene are synthesised
- the short DNA fragments are joined together by DNA ligase
What are the advantages of using a gene machine?
-quick
- the most accurate method
- intron free
What is cDNA?
complimentary DNA
What needs to be at either end of the isolated gene for the desired gene to be transcribed?
the promoter region at the start = so DNA polymerase is stimulated
a terminater region at the end = DNA polymerase is released to end transcription
How is a vector formed from a plasmid so desired genes can bind?
- plasmid is cut by restriction endonucleases - the same ones that were used to isolate the desired gene
- DNA ligase splices the desired genes to the plasmid sticky ends by complimentary base pairing = forms recombinant plasmid
- some ordinary plasmids are reformed as their sticky ends with join back together
Why are the same restriction endonucleases used for plasmids and donor DNA to form a recombinant plasmid?
so the sticky ends on the plasmid vector are complimentary to the sticky ends on the recombinant DNA produced from donor DNA
so that they can complimentary base pair to form a recombinant plasmid
Do bacteria take up a lot of little plasmids?
little amount of plasmids uptaken
How are plasmids reintroduced to bacterial cells?
mix plamids with the bacterial cells - the bacteria will take up a little amount of regular and take up even less recombinant plasmids
How are bacteria containing the recombinant DNA identified?
using genetic markers
What are the 3 diff types of genetic markers used to identify recombinant plasmids?
- genes for antibiotic resistance
- genes that code for flouresence from jellyfish
- gene that codes for an enzyme that has an action that can be identified eg, colour change/ change in pH
What are genetic markers?
any gene that is easily identifiable
What are the 2 antibiotic resistance genes examples?
Tetracyline and Ampicillin
How is a recombinant plasmid formed from a plasmid with the 2 resistance genes for identification?
- the plasmid is cut by restriction endonucleases in the middle of the Tetracyline resistance gene = disrupts the tetracyline resistance
- bacteria are only resistant to ampicillin now
- the sticy ends on the desired gene complimentary base pairs with the sticky ends on the plamid
- desired gene is inserted in the middle of the tetracyline resistance gene
- some of the original plasmids reform
What are the 3 diff types of bacteria produced by using antibiotic resistance as a genetic marker?
- bacteria w/ no plasmids = no uptake - no resistance
- bacteria w/ recombinant plasmid = resistanct to only ampicillin
- bacteria w/ original plasmid = resistance to both tetracyline and ampicillin
Describe the process of replica plating to identify recombinant plasmids
- create a master plate containing all 3 diff types of bacteria
- press down lightly on the master plate using a muslim cloth to pick up small amounts of bacteria from their colonies
- transfer these bacteria to an ampicillin agar plate, = press down on the plate with the muslim cloth
- repeat by transferring bacteria from master plate to a tetracyline plate
- colonies of resistant bacteria will form relevant to the antibiotic used
Which type of bacteria is the recombinant plasmids - the bacteria you need to identify containing recombinant DNA?
the bacteria with plasmids resistant to ampicillin but not resistant to tertacyline
How are fluorescent markers used to identify recombinant plasmids?
use the GFP = green flourescence protein
insert the donor gene in the middle of the GFP gene to disrupt the production of GFP
looking for the bacteria that do not fluoresse
What enzyme marker is used to identify recombinant plasmids?
lactase
How is lactase used as an ezyme marker to identify recombinant bacteria?
lactase breaks down lactose and turns the colourless products to blue
recombinant DNA is added to the middle of the lactase gene so the gene is disrupted
the recombinant DNA is colourless
What happens to the bacteria while they are culturing?
binary fission = bacteria replicate so they are all genetically identical
What is a colony?
a large group of bacteria which are all genetically identical
What are the 2 different methods to amplify DNA fragments called?
in vivo
in vitro
What is in vivo cloning?
any work that is done inside of the living org
What is in Vitro cloning?
any work performed outside of the org
What is the process of in vivo cloning?
recombinant DNA process
restrictuion endonucleases
insertion in vectors
How does DNA ligase join together DNA fragments?
catalyses condensation reaction to form phosphodiester bonds b/w nucleotides
What is the name of the method for in virto cloning?
PCR - polymerase chain reaction
What is DNA amplification?
cloning so there is an exponential increase in the number of DNA molecules
What does exponential mean?
doubling per unit of time/ per cycle
What is added to the start of the PCR?
DNA with desired gene
DNA nucleotides
DNA polymerase
primers
Describe the process of the PCR to amplify DNA fragments
- add DNA polymerase, DNA nucleotides and primers to the thermocycler
- original DNA is denatured by heating to 95 degrees, separating 2 strands of DNA
- solution is cooled to 55 degrees triggering primers to anneal to each of the single strands of DNA by CBP
- solution is heated to 72 degress which is the optimum tmep for taq polymerase = catalyses the synthesis of a complementary strand for each single strand of DNA by joining nucleotides
- the process is repeated to double the quantity of DNA produced each time
What does it mean if DNA has been denatured?
H bonds have been broken between DNA strands
What does anneal mean?
complementary base pair and bind
What is a thermocycler?
a machine that controls changes in temp
What is taq polymerase?
DNA polymerase from a thermophilic (extremophile) bacteria that lives in hot temps
it acts the same as DNA polymerase
What is the optimum temp for taq polymerase?
72 degrees
What are primers?
short sequence of single stranded DNA - complimentary to start and end of DNA sequence being cloned
What is the role of primers?
they signal to DNA polymerase to start synthesising and replicating DNA strands
What are the advantages of using primers in PCR?
- stimulates DNA polymerase for cloning and replication
- prevents DNA strands CBP to each other and keeps them as single stranded DNA
What are the 5 limitations of PCR?
contamination
highly sensitive to inhibitors
error rate
size of DNA copied
limit to how much DNA can be amplified
What is the issue of contamination in PCR?
any DNA in the machine will be amplified so if there is any contamination from reagents, not cleaning the machine properly or the experimenter, this will also be amplified
Why is it an issue that PCR process is highly sensitive to inhibitors?
molecules in solution may inhibit enzymes or primers annealing
How does PCR have a high error rate?
mutations occur and cause mistakes in replication
taq polymerase does not have a proof reading ability to identify errors in base seq
PCR cycle continues and error rate increases
In what type of cloning does DNA polymerase have a proof reading function?
in vivo cloning
Why is the DNA that is copied from PCR relatively small?
the machine can’t read more than 3000 base pairs and human genes are a lot larger than this so it is difficult to read all of the gene
copying is only effective up to 3000 BP
How many BP is in vivo cloning effective up to?
2 million BP
What 3 factors may limit how much DNA can be amplified in PCR? (factors that limit PCR)
- ran out of nucleotides to synthesise new DNA strands
- taq polymerase starts to denature
- DNA strands rejoin together insteads of primers annealing
For which cloning tech requires culturing techniques?
in vivo cloning
What will happen as soon as the DNA is inserted into bacteria in in vivo cloning?
once the DNA is in bacteria, DNA will automatically be copied
What does in vitro cloning require for copying to occur?
the correct primers
Which cloning process is more rapid?
in vitro cloning
Which cloning tech is more accurate?
in vivo cloning
Where does DNA need to be insterted to synthesis the gene for in vivo cloning?
transformed bacteria can synthesise the gene product
Where does DNA need to be insterted to synthesis the gene for in vitro cloning?
gene must be inserted into a cell to synthesise the gene product
What are the medical benefits of recombinant DNA tech?
able to synthesise human products for example insulin to treat Type 1 diabetes
What are the agricultural benefits of recombinant DNA tech?
can change plant traits for more plant growth
eg:
disease resistance
nutritional prospects
toxicity to pests
What are the ecological benefits of recombinant DNA tech?
GM bacteria can remove impurities from the environment
What is added to a DNA fragment before it can be transferred to a host cell?
promoter region and terminator region
What are the conditions to transform host cells?
bacerial cells and plasmids can be added to ice-cold calcium chloride solution and the temp is increases to 42 degress for 2 mins to heat-shock the bacteria
What is a gene probe?
a short segment of DNA that has complimentary bases to the desired gene. it is labelled with fluoresence or radioactively labelled
What are gene probes used for?
used to identify certain alleles
used to screen pateints for hertiable disorders, response to drugs and health risk
How is a gene probe produced and used to identify a mutant allele?
- base seq of mutant allele of the gene needs to be determined
- DNA fragment w/ complimentary base seq for mutant allele is produced
- DNA fragment is fluoresently/ radioactively labelled to form a gene probe
- DNA probe is amplified by PCR
- DNA probe is added to single-stranded patient DNA and probe will anneal to the mutant allele if present
- Patient’s DNA is labelled with DNA probe
- exposure to UV light (flouresence) or X-ray (radioactive) the mutant allele can be identified if it is present
Why would a delay in the insertion of a desired gene produce orgs without the desired gene?
cell division has occured
orgs gametes do not recieve the gene
What is the role of restriction endonucleases when insterting DNA fragments into plasmids?
to cut the plasmid
What is the role of DNA ligase when inserting DNA fragments into plasmids?
joins the DNA fragment to the plasmid
What is the role of taq polymerase in PCR?
joins nucleotides to produce complementary strands of DNA
Why would a DNA sample be hydrolysed before added to PCR?
to remove any DNA present
this DNA would be amplified
What is the issue with using restiction enzymes to insert a fragment of DNA from a human into bacteria?
Human DNA contains introns
bacteria cannot remove introns
What is the advantage of using GFP (fluoresence) to identify bacteria that have taken up plasmids?
can quickly identify transformed bacteria using UV light
Why would the desired gene be injected into gametes instead of full orgs?
the gene can get into all or most cells
so that most cells have the desired characteristic
What are VNTRs full name?
variable number tandem repeats
What are VNTRs made up of?
introns - non-coding repeated sections of DNA bases
How can you tell if 2 people are more related from VNTRs?
they will have more similar VNTRs
How are VNTRs cut out of genes?
using restriction endonucleases cut at recognition sites
What are the uses of genetic fingerprinting?
determine genetic relationships and variability, taxonomy relatedness
paternity tests
medical diagnosis
foresensic science
What is genetic fingerprinting?
comparing VNTRs via electrophoresis
Describe the process of gel electrophoresis
- DNA has been extracted from bio materia and amplified if nescesary
- fragments are loaded into wells containing agar gel at one end (negative charged end),
- a voltage is applied to the gel and DNA fragments move towards the positive charge
- alkaline is added to gel to separate DsDNA to single stranded
- A nylon membrane is added to the agar well to pick up fragments from each band
- DNA bands are transferred onto the nylon membrane
- labelled DNA probes are added and CBP w/ VNTRs present
- wash away any unbound DNA probes
What are the pros of using genetic fingerprinting?
- non-invasive
- can use really small sample that would be too small for other tests like a blood test
- reverse wrongful convictions
- store genetic info in a database on isolated populations around the world before they intermix and are lost
What are the issues of genetic fingerprinting?
- violation of privacy
- genetic info stored on a computer database which is vulnerable to hacking and missuse
- there are rigth regulations of who has access - insurance companies or buisnesses may have access
- genetic profiles offer probabilities that are not absolute
- may result in wrongful convictions and be used to influence the jury
What is genetic screening?
using DNA probes to test for genetic disorders from patients single-stranded DNA
What are the 2 uses of genetic screening?
personalised medicine
genetic counselling
What is personalised medicine?
providing health advice or medication that is based on an individual’s genotype
What is genetic counselling?
giving advice and information about certain disorders you may be predisposed to found by genetic screening
What are 2 positives of personalised medicine?
avoids false hopes for cure and treatment
treatment is more effective and more cost-effective as you know the exact dosage and not over-prescribing
What are the good things for genetic screening?
people who have the genes for certain genetic disorders can have additional screening for earlier detection
ppl can chane lifestyle choices to reduce risks of disorders
What are the issues with genetic screening?
just bc you have the gene doesn’t mean you develop the disorder - probabilities and not absolute, may cause unescesary stress
you can develop disorders without the genes
Why do DNA fragments move towards the positive charge on gel electrophoresis?
they have a slight negatie charge due to phosphate groups
Which type of DNA fragments move faster and further on the agar in gel electrophoresis?
shorter DNA fragments
Why are the DNA fragments prodcued for gel electrophoresis different sizes?
restriction endonucleases cut DNA at randomly spaced recognition sites
What is a DNA ladder?
adding fragments of DNA that have known lengths to gel electrophoresis
How do fragments separate in gel electrophoresis?
according to length
What is DNA hybridisation?
heating double-stranded DNA to break it down into single stranded DNA
and mix with DNA probes to CBP
How would scientists see if a cell contains a certain gene using a gene probe?
- extract DNA and add restriction endonucleases
- separate fragments using electrophoresis
- treat DNA to form single strands and expose bases
- The probe will bind to the desired gene
Why is DNA on the nylon membrane treated to form single strands?
so that gene probes can bind
What is the use of a DNA ladder?
has DNA fragments of known sizez to compare the position of other fragments
Why is it important to identify the specific strain of bacteria infecting a patient?
- to see if the strain is resistant to any antibiotics
- so they can presecribe the right antibiotics
