6.1.3 Manipulating Genomes Flashcards
what is SD’s - PAGE Electrophoresis
method used to seperate proteins
what can electrophoresis be used for
to analyse mixes of proteins to determine what is present
What is DNA profiling
short tandem repeats are compared as people sharing an str is very low
What is a short tandem repeat
repetitive sequences of 10 -100 base pairs
Steps of DNA fingerprinting
Extract DNA
cut DNA using resitriction enzyme
seperate DNA fragments using electrophoresis
Then do the same with the individual being compared
How to do southern blotting
-cut DNA out of gel and purify it
- or place nitrocellulose sheet over gel and press down leave over night and the DNA transfers to the sheet
What is a probe in Southern Blotting
single stranded and complementary to DNA your trying to find
What does a probe do
bind to DNA its complementary to and lets out a fluoresent colour to identify it easily
How are probes labelled
radioactively or with fluorecent colour
DNA Sequencing definition
what order ATCG go
Applications of DNA Sequencing
identify mutations
identify genetic disorders
used to help create vaccines
identify common ancestors
prove paternity
find desired characteristics and genetic engineer them
Sanger sequencing method
4 test tubes
put fragmentated DNA to be sequenced, bases(A,T.C.G), DNA polymerase and primers into all the test tubes
Add modified bases (ddNTPs) which are radioactive- one for each testube
What happens during sanger sequencing
DNA polymerase adds bases onto primer and produces many new strands
Everytime a ddNTP is added it terminates the chain so fragment ends
High throughput sequencing
first automated DNA sequencing machine was made in 1986
used fluoresence rather than autoradiography
Bioinformtives
develops and uses computer software to analyse, store and organise biological data
What is the data in bioinformatives
univeral
shared internationallly
DNA, RNA, Protein
Genome
total complement of an organisms genes
what does Dna sequencing do
determines the order of nucleotides in a dna sample
common techniques of gene technologies
restriction endonucleous
PCR
Electrophoresis
Dna probes and microarrays
dna profiling
dna sequencing
genetic engineering
gene therapy
DNA Fragmentation method
Restriction endonucleases recognise palindromic sequences and cut the dna and these sites
leaves overhanging sticky ends
each enzyme is specific to a sequence and is complementary to their active site
what does PCR do
makes more copies of a section of DNA
amplifies the dna
Uses of PCR
identify viral rna/dna in patient
forensics
ancient organisms
epidermiology
detect and identify mutations e.g cancerous mutations
PCR mixture
sample of dna
excess of 2 primers
dna polymerase
4 dna bases
appropriate buffer
4 steps of PCR
denaturation
annealing
elongation
amplification
What happens in the 1st step of PCR
denaturation
DNA is heated at 95 degrees to break hydrogen bonds between bases to get a single strand
what happens in the 2nd step of PCR
annealing
cool to 68 degrees
primers will anneal to dna by complementary base pairing
what happens in 3rd step of PCR
elongation
heat to 72 degrees to allow dna polymerase to extend the primers and copies of the target dna are synthesised using target strands as templates
what happens in the 4th step of PCR
amplification
each time the above 3 steps are repeated the number of DNA strands doubles
what does comparitive gene mapping do
identifies importance of difference genes
can see how tiny changes in gene causes different features
comparing species identifies evolutionary relationships
synthetic biology
designs and builds biological devices and systems
why is knowing the dna sequence good
can lead to modelling the amino acid sequence
examples of synthetic biology uses
synthetic new proteins
biosensors
nanotechnology
storing information
new medicines
genetic engineering definition
direct manipulation of an organisms genes
what can genetic engineering involve
addition of genes
removal of genes
silencing of genes by blocking genes expression
what is dna ligase used as
a sealing enzyme
what is a dna probe
short single strand dna fragment
labelled with 32P or fluoresence
used to locate a gene
Method to genetically engineering an insulin gene
Cut out insulin gene using restriction enzyme and leave sticky ends
cut plasmid from bacteria with the same restriction enzyme
insert gene into plasmid using DNA ligase
transgenic bacteria reproduce in a fermenter
genetically engineered insulin is then produced by the bacteria
Why do you use the same restriction enzyme to cut the plasmid and the insulin gene
so they have complementary sticky ends that overhang
what enzyme is used to insert insulin gene into the plasmid
DNA ligase
Stages of genetic engineering
obtain gene of interest
insert gene into vector
insert vector into cell
or directly insert gene into the cell
How do you obtain the gene of interest if you don’t know the sequence of gene
extract mRNA
use reverse transcriptase to make single stranded cDNA
then use DNA polymerase to make it double stranded
How do you obtain the gene of interest if you know the sequence of the gene
can be produced using automated synthesiser
amplify gene from genomic DNA using PCR
can cut it out of genomic DNA using restriction enzyme
Types of vectors you can insert a gene into
seal in a virus
use a bacterial plasmid using same method as insulin
ways to insert the vector into a cell
heat/cold shock
electroporation
electrofusion
transfection
T1 plasmid
Differences between somatic and germ line gene therapy
Somatic is body cells Germ line is gametes
Somatic cures you germ line cures your offspring
Somatic causes specific cells to have gene germ line all cells have the gene
How does inserting a new gene into a chromosome could affect the functioning of other genes in that chromosome
Gene expression changes
Newly genes switched on or off
New gene could disable functioning gene if its inserted
GM crop characteristics
Good growth conditions
Pathogen resistance
Pesticide resistance
Herbicide resistance
Nutritional value and good appearance
Long shelf life
Pros and cons of good growth conditions for GM crops
High yield
Expensive
Pros and cons of pathogen resistance for GM crop
High yield
Transfer of gene to wild species
Pros and cons pesticide resistance of GM crops
High yield helps poor farmers
Biaccumalate pests can become resistant
Pros and cons of herbicides resistance for GM crops
High yield
Create superweeds which are resistant to herbicides
Pros and cons of nutritional value and appearance for GM crops
More likely to be brought
Good for human health
Medicine development
May be allergenic for some people
Pros and cons of long shelf life for GM crops
Easy to import and export
Better customer satisfaction
Commercial value decreases as people don’t need to buy them as often
Gene therapy
Involves treating genetic diseases by altering a patients natural genotype
How to do gene therapy for somatic cell therapy
Normal allele is incorporated into a loop of dna which is attached to liposomes
Liposome-DNA complex is sprayed as an aerosol of fine droplets into nose of cystic fibrosis sufferers
Advantage of somatic gene therapy
Better quality of life
Removes symptoms
Disadvantages of somatic gene therapy
Not a cure
Has to be done on a regular basis
Pass on abnormal gene to offspring
Advantage of germ line gene therapy
Cure for children / offspring
Disadvantages of germ line gene therapy
Not a cure for self
Expensive as it’s ivf
Pharming
Using GM animals to produce pharmaceutical
What happens in pharming
Add or remove genes so the animal developers certain discuss to act qs models in development of new treatments
Example of pharming
Knock out mice have genes deleted so they develop cancer
Action of a restriction enzyme
Cuts the dna into fragments at a certain recognition site leaving sticky ends
2 ways in which bacteria which take up modified plasmid can be identified
fluorescent substance and which ever plasmid is fluorecent contain it
antibiotic resistance gene introduced and survivors have plasmid
restriction enzyme
cuts dna at specific site leaving sticky ends
characteristics of restriction enzymes target site
palindromic
pairs
specific sequence
advantages of obtaining human insulin by genetic engineering
faster production
less chance of it getting rejected
easier to grow on mass scale
3 reasons researchers may be more concerned about potential risk of dna vaccines compared with protein based vaccines
could trigger autoimmune response
could undergo mutations
create new disease
what is recombinant dna
dna from 2 differnt dources joined
