DNA Technology Flashcards
How do you isolate genes
Use enzymes Either Reverse transcriptase Or Restriction endonuclease
How is reverse transcriptase used to isolate genes
Locate and extract gene
mRNA carries the code
mRNA acts as template for cDNA
cDNA formed using RT enzyme
DNA polymerase binds complementary nucleotides on cDNA template
Double stranded DNA formed using cDNA template
Copy of desired gene in DNA
How is restriction endonuclease used to isolate genes
Cuts DNA at recognition site
Cut leaves sticky ends so bigger bonding site
Complementary bases pair up
Enzyme DNA ligase joins them
What is In Vivo gene cloning
Transferring fragments to a host cell using a vector
What ways can DNA be inserted
Using plasmid vector
Using virus
Coat in lipids
What is the role of a vector during in Vivo gene cloning
Transfers genes from one organism into another
What vector is used to insert DNA into host cell
Plasmid
What is transformation
Reintroducing the plasmid back into bacterial cells
Describe the process of transformation
Mix together in medium containing calcium ions
Calcium ions and temperature change
Causes bacterial cells to become permeable
Plasmid passes through membrane into cytoplasm
Why do not all bacteria cells have the DNA fragment
Only a few take up plasmids when mixed together
Some plasmids close up before DNA fragment is incorporated
How do you identify which host cells have taken up the gene
Enzyme gene marker
Fluorescent gene marker
Replica plating using antibiotic resistance markers
What happens when doing antibiotic resistance markers
Use plasmid with 2 antibiotic resistant genes
Insert desired gene into bacteria
Do replicate plate
Put one plate with disease
If survive, shows resistant to gene which shows did not take up wanted gene
Ones we want die so know which ones on non-replica plate to keep and grow
How are fluorescent gene markers used
Jellyfish have green fluorescent protein
Transfer desired gene into plasmid from jellyfish
If bacteria cell taken up gene then they won’t fluoresce when viewed under microscope
How are enzyme gene markers used
Gene marker codes for enzyme eg lactase
Lactase turns substance from clear to blue
Desired gene placed in centre of lactase gene
If gene present, won’t cause colour to change to blue in presence of lactose
Why are fluorescence and enzyme gene markers more efficient than antibiotic ones
Won’t kill ones we want
So it’s quicker method
When growing bacteria, what needs to be controlled
Temperature
PH
What is In Vitro gene cloning
Polymerase chain reaction
What is a thermocycler
Controls temperature changes
What is polymerase chain reaction used for
Crime scene investigators
Info gaining about long dead organisms
Determine to identify father
What are primers used for
Keep strands apart
What is DNA polymerase used for in polymerase chain reaction
Join together nucleotides
In Vitro
How are strands broke apart
Temperature
Break hydrogen bonds
In Vitro
Why is heated to 95 degrees
To spear ate DNA strands
In Vitro
Why is cooled to 55 degrees
Allowed primers to anneal
And primers to attach to start and end complementary bases on DNA strands
In Vitro
Why is heated to 72 degrees
Optimum temperature for DNA polymerase
Describe polymerase chain reaction process
Put in thermocycler Heated to 90 degrees to break bonds Cooled to 55 degrees So primers can be used Heated to 72 degrees Nucleotides bind to complementary bases DNA polymerase
Now have 2 DNA molecules
Cycle takes 2 minutes
Cycle keeps going
What are the 5 main stages of DNA technology
Isolation Insertion Transformation Identification Growth/cloning
Advantages of In Vivo
No risk of contamination
Very accurate
Makes copies of specific gene not whole DNA sample
Disadvantage of In Vivo
Takes longer than In Vitro to produce same quantity
Advantages of In Vitro
Doesn’t require living cells
Very quick process
Disadvantage of In Vitro
Requires very pure sample as could contaminate with other DNA, leading to a false result
Benefits of genetic modification
Increase yield from crop Improve nutrient content in food Introduce resistance to disease Making vaccines Produce medicine for treating diseases Develop tolerance to environmental conditions
Describe process of Anti-thrombin production
Egg and sperm
Put gene in fertilised egg before division occurs
Offspring with gene are crossbred
Extracted and purified
Benefits of recombinant DNA technology
Genetic fingerprinting used in forensic science
Gene therapy to cure genetic disorders
Genetically modified crop has economic and environmental advantages
Risks of recombinant DNA technology
Don’t know long term effects
Gene may pass to different organisms, causes problems
What is genetically engineered gene mutates?
Expensive. Money better use eg fighting world hunger
how can cystic fibrosis be treated using gene therapy
gene replacement
gene supplementation
germ-line gene therapy
somatic-cell gene therapy
what is germ-line gene therapy
replacing defective gene in fertilised egg so cells of offspring will develop normally
what 2 methods are used to deliver genes in gene therapy
harmless virus (adenovirsues) wrapping it in lipids
both then inserted in patient’s nostrils
why are forms of delivering gene therapy not always effective
adenoviruses may cause infections
patient may develop immunity to adenoviruses
even if delivered successfully, few genes actually expressed
How would you use gene therapy to treat severe combined immunodeficiency
Normal enzyme isolated from healthy human tissue
Then inserted into retrovirus
Retrovirus mixed with patient’s T cells
Retrovirus inject copy of normal enzyme gene into T cells
T cells reintroduced into patient’s blood, now with genetic code needed to make enzyme
What is somatic-cell gene therapy
Gene therapy that targets just the affected tissue
Healthy genes are not passed on to future generations
Means treatment needs to be repeated
