21. recombinant DNA technology Flashcards
Five stages of DNA Technology
- isolation of DNA for a specific protein (creation of DNA fragment)
- insertion of DNA into a vector
- transformation of DNA into a host
- identification of successful hosts
- cloning/growth of host cells
Describe how reverse transcriptase can be used to isolate a DNA fragment.
Enzyme that uses mRNA as template and joins DNA nucleotides, resulting in single-stranded cDNA,
+ DNA polymerase = DNA (H-bonds)
What are three way that DNA fragments can be isolated?
Reverse transcription
Restriction endonuclease
Gene machine
Describe how a gene machine can be used to create DNA fragments
Machine that works out mRNA and DNA sequences from amino acids.
Sequence is checked for biosafety and biosecurity, ensuring that gene is safe and ethical to produce
Results in oligonucleotides
What are oligonucleotides and what makes them?
small sections of overlapping, single stranded nucleotides
produced by gene machine
Describe how restriction endonuclease can be used to produce a DNA fragment.
- recognition sites bind the enzyme to specific areas on DNA base sequence
- results in staggered/sticky ends
Give an advantage and disadvantage of using reverse transcriptase.
Advantage
- lots of mRNA of interest form selected cell
Disadvantage
- time consuming
Give an advantage and disadvantage of restriction endonuclease.
Advantage
- sticky ends make insertion easier
Disadvantage
- still contains introns
Give an advantage and disadvantage of gene machine.
Advantage
- quickly makes exact fragments of DNA with sticky ends
Disadvantage
- need to know sequence of amino acids beforehand
Why might host cells not get transformed?
- Plasmid rejoins before DNA fragment could bind
- Plasmid does not enter host cell
What is the difference between in vivo and vitro cloning?
In vivo involves use of a vector to clone recombinant DNA
in vitro (PCR) doesn’t.
Describe how DNA is inserted into vector.
- plasmid inside vector is cut using same reverse transcriptase that cut DNA fragment.
- results in complementary sticky ends between the DNA fragments.
- the enzyme ligase aneals them together. (= phosphodiester bonds)
What is a plasmid?
Circular loop of DNA, found in bacteria, separate from rest of genome
How is the host cell ‘transformed’ with a vector? (in vivo cloning)
- membrane of host cell is made more permeable by mixing with Ca+ and heat shocked
- vector can now diffuse across membrane
What are three types of marker genes that can be used to identify transformed host cells?
- antibiotic resistance genes
- fluorescent gene markers
- Enzyme markers.
How are DNA fragments (made by restriction endonuclease) modified?
Promoter region added to the start of DNA. This is the binding site for RNA polymerase to enable transcription
Terminator region added to the end of gene. causes RNA polymerase to detach, ending transcription between genes
Explain how antibiotic-resistance genes can be used to identify transformed cells.
Plasmid has genes for the resistance of tetracycline and ampicillin.
Insertion of DNA fragment disrupts resistance to tetracycline.
Use a sterile stamping block to transfer colonies of bacteria onto agar plate with ampicillin.
repeat onto tetracycline
cultures that grow on ampicillin have successfully taken in plasmid, cultures that don’t grow on tetracycline have taken up DNA fragment. (=clone from original plate)
How can fluorescent markers be used to identify transformed cells?
green fluorescent protein (GFP) gene inserted to plasmid.
DNA fragment disrupts GFP gene and prevents production
non-glowing/ fluorescent colonies contain recombinant plasmids
How can enzyme markers be used to identify transformed cell.
The gene for the enzyme lactase inserted into plasmid, disrupted by DNA fragment.
colonies grown on colourless substance (blue if lactase gene not disrupted)
Explain the PCR method.
The temperature increased to 95C to break H bonds between DNA.
temperature is then decreased to 55C, so primers can attach (annealing)
taq DNA polymerase then attaches free nucleotides and makes new strand. (synthesis) the temperature is increased to 72 (optimal for taq DNA polymerase.
three advantages of PCR
automated, efficient
Rapid,
no living cells, less complex + quicker
difference betwen taq polymerase and normal DNA polymerase
bacterial protein that can withstand high temperatures (used in PCR)
what are DNA probes?
what are they used for?
labeled single-stranded pieces of DNA (fluresc or radio)
locate specific gene location and screen for heritable conditions
How are DNA probes used?
sample of patient DNA is heated to make it single-stranded
it is mixed with DNA proves that are complementary to a range of genes and alleles (eg for diseases)
if patient has that allele, DNA will bind with this probe, identified with x-ray or UV
…or a specific allele (for specific disease)
what is DNA hybridisation?
How is this used?
DNA is heated to separate double helix into single strands. mixed with complementary single stranded DNA
once cooled, complementary strands anneal.
find particular allele (with DNA probes) used to select medicines based on genotypes. (some drugs more useful dependant on genotype)
what is genetic counselling?
advice based on screening results of disease causing alleles.
eg lifestyle, monitoring.
what are VNTRs
how can they be used?
variable tandem repeats, repeated sequences in introns. closely related = similar VNTRs.
genetic fingerprinting studies VNTRs, to determine genetic relationships and variability.
Briefley explain 7 steps of genetic fingerprinting,
Collection/extraction, digestion, seperation, hybridisation, development, analysis
Collection
eg crime scene
Extraction
PCR (if sample size is small)
Digestion,
restriction endonuclease cuts out VNTRs
Separation, (gel electrophoresis)
placed on polar gel negative DNA moves to the positive end of gen. smaller pieces travel further due to resistance of gel.
Hybridisation,
DNA probes added, complementary to VNTr
Development
probes transferred to nylon sheet, were they can be exposed to UV or X-ray
Analysis
How can DNA fingerprinting bands be interpreted?
when might you use it?
bands compared to different organisms, most matching/ similar bands are more likely to be unknown sample
paternity, crime scene, medical diagnosis, avoid inbreeding
define genome and phenome
genome is complete set of genes in cell, phenome is all the proteins that a cell is able to produce
