dna manipulation Flashcards
what are endonucleases?
enzymes that can break the sugarphosphate bonds between nucleotides of nucleic acids
- cuts dna
scissors
what are restriction enzymes?
type of endonuclease produced by bacteria
- cut dna at their specific sequences/recognition site
why is it useful to cut with the same restriction enzymes?
- will be specific to one sequence/has unique recognition sites
- plasmid and target gene cut sites will have complementary nucleotides overhanging
- attracted to each other= more likely target gene will insert in the plasmid
what are sticky ends?
a way in which dna is cut leaving overhanging unpaired nucleotides
exposed bases able to join to other dna with complementary sticky ends
what are blunt ends?
no overhanging nucleotides
what is dna ligase?
enzymes that join nucleic acid fragments together
- creates the bods between sugar and phosphate to join dna
glue/tape
what are polymerases
enzymes that join nucleotides together to create nucleic acids
- synthesises dna/amplifies fragments of dna
what is the function of crispr cas9 in bacteria
help them fight off viral infections
- target and cut viral dna= protect the cell
outline the process of how bacteria uses crispr
- virus injects viral dna
- viral dna added to bacterial genome
- guide rna formed complementary to viral dna
- grna guides cas9 to target gene and destroys viral genome
what is cas9 and its role
- endonuclease that carries a piece of rna inside it
- sequence of dna found is complementary to the guide rna
- cas9 cuts
how can scientists use crispr cas9
programmable
- identify the desired dna sequence
- crispr cas9 can be directed to it
what is the role of sgrna/grna
guides cas9 and tells it where to cut
grna: bacteria. sgrna: made by scientists
how is cas9 programmed
changing sgrna
- identify desired sequence
- create sgrna made complementary to that target gene
- sgrna joined to cas9
- = cas9 now programmed
what is the pam sequence and its role?
very short sequence of nucleotides on dna
- signals cas9 to stop and check for complementary dna sequences to cut
no pam sequence=no cut=cannot be modified
benefits of the pam sequence
- efficient: cas9 only looks for pams instead of searching through and unwrapping every dna sequence
- protect bacteria dna: bacteria never have pam sequence in their own dna= dna cannot be cut up
2 ways crispr can be used to create genetically modified organisms
after cas9 cuts dna:
- gene knock in “editing”
- gene knock out “silencing”
= new nucleotides can be added to repair or silence target gene
what is gene knock in
editing
new dna sequence is inserted into the dna break
- allows faulty gene sequence to be replaced with correct sequence
= restore normal gene function
what is gene knock out
silencing
insertion/deletion of bases
- changes the way nucleotide sequences is read
= disables gene function or producing STOP signal
= silences faulty gene
technological uses of crispr?
- correcting mutations responsible for disease
- switching faulty genes off
- adding new genes to an organism
- studying the effects of specific genes
what is amplification
creates many copies of an original dna sample
repeat process many times
what is polymerase chain reaction?
dna amplification technique
- rapidly makes many copies of an original dna sample
2^x
x= number of cycles
what is the purpose of amplification
increase quantity of identical copies of dna available
- large enough sample to be analysed
what components are needed for pcr?
- dna sample
- dna polymerase (taq)
- dna nucleotides
- primers
- mix buffer
- pcr tube
- thermal cycler
rna converted to dna- enzyme req for amp acts on dna,
define target gene
gene of interest
- gene we want the bacteria to express
cut out of genome
define plasmid
ring of dna
what is a plasmid vector?
circular ring of dna
- used as a means of transporting the gene of interest into the bacteria
what is a recombinant plasmid?
plasmid that has had the gene of interest inserted into it
how is a recombinant plasmid created?
- restriction enzyme cuts plasmid
- same restriction enzyme removes target gene from dna
- target gene and plasmid have complementary sticky ends
- ligases join two pieces of dna
complementary sticky ends= easier to join
explain the process of bacterial transformation with antibiotic resistance gene
- gene of interest generated, inserted in appropriate bacterial plasmid vector chosen
- bacteria are mixed with plasmids
- heat/electroshock to encourage bacteria to take up recombinant plasmid
- bacteria that take up recombinant plasmid= transformed
- bacteria cultured on antibiotic medium
= culture only transformed bacteria that will express gene of interest= can grow and form colonies - untransformed bacteria die
eg. gene for dystrophin is cut from human dna
- plasmid vectors have antibiotic resistance gene
what is the purpose of bacterial transformation? how can plasmids be used to make bacteria produce stuff
genetically modify bacteria to synthesis large amounts of protein
- transformed bacteria are grown/cultured and will transcibe and translate [gene of interest] producing [gene] which can then be harvested
how could you use bacterial transformation to produce human insulin?
- forgien dna and plasmid with amp resistance gene are cut in the middle of lacZ gene= produces b-galactosidase
- bacteria cultured on a plate w amp and substance that changes colour when exposed to b-galactosidase
- some cells do not take up plasmids= not amp resistant= dies/killed by amp
- bacteria take up nonrecombinant with intact lacZ gene= survive antibiotic+changes colour
- bacteria takes up recombinant plasmid= can’t produce b-galactosidace= survives+lacz no function= white colonies grow
lacZ: where restriction site is
what is a reporter gene
- distinguish between recombinant and non-recombinant plasmids
- genes with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest
eg. recognisable flourescent protein
what are ways to use reporter genes to recognise bacterial transformation
reporter gene continuous: completely expressed
- bacteria that have been transformed with non recombinant plasmid to glow under UV
reporter gene noncontinuous: split by gene of interest
- bacteria taken up recombinant plasmid= cannot glow
what are antibiotic resistance genes?
- distinguishes between transformed and untransformed bacteria
- only transformed bacteria contain the gene for antibiotic resistance= survives and multiplies
eg. amp^r confers ampicillin resistance
what are the steps and temps of pcr
denaturing: 95°
- strands seperate
annealing: 55°
- primers bind template
extending/elongation= 72°
- synthesis new strand
describe the process of denaturing
-dna is heated to 95°
- breaks hydrogen bonds from heat
- 2 halves seperate= forms single stranded dna
describe annealing in pcr
- sample cooled to 55°
- allows primers to bind to complementary sequences of single stranded dna
what is a primer and its purpose (why are 2 needed)
short complementary single strand of nucleic acid
- acts as a starting point for dna polymerase to begin building dna strand
- provides taq polymerase with binding site to begin synthesis
(nucleotide sequences at 5’ end of the coding and template stands are different)
what is elongation/extending in pcr
- dna heated to 72°= taq polymerase work optimally
- binds to primer
- begins synthesising a new dna strand using complementary base pairs
what is taq polymerase and why is it useful for pcr
type of dna polymerase
- does not denature at high degrees-> can maintain its shape and function at up to 95°
- optimal temp is high (72°)
what is gel electrophoresis
technique used to compare dna samples by separating dna fragments according to their size
what are the components of gel electrophoresis
- dna samples placed in wells (negative electrode)
- first well contains sample with dna fragments of known size (dna ladder)
- electric current seperate the fragments
why does dna from each sample appear in a series of bands on the gel
- restriction enzymes cut the dna at a specifically recognised sequence
- different individuals have different dna sequences= restriction enzyme will cut the dna in different places
=> results in fragments of varying size
what direction does dna move through in gel electrophoresis
dna has overall negative charge= attracted towards + electrode, repelled from - electrode
factors that affect the rate of movement of dna fragments through agarose gel
- size/length of fragment (smaller dna fragments move quicker= travel further)
- conc/viscosity of agarose
- voltage/power used
(NOT LENGTH OF TIME)
what is the benefit of having degenerate codons
degree of redundancy
- chbaged to original dna sequence through mutations may not lead to insertion of different amino acid
benefit of 2 types of genes onplasmid
another layer of selection
- let us know if bacteria is successfully transformed
gmo def
genetically modified organism
- organism that has had its dna artificially altered in any way
tgo def
transgenic organism
- organism that has had a gene from a different species inserted into its genome
(specific type of gmo)
benefits of gmo?
- increase crop yield= more nutritional value, store longer
- decrease in pesticides, herbicides
- drought, salt tolerance
- improvement in health of humans, medicines
- reduces cost and time associated with crop loss
risks of gmo?
- possible uncontrollable spread pf trans genes into other species
- release of gmos into environment may be irreversible
- animal welfare and ethical issues: poor health and reduced lifespan
- emergence of pests, insects, microbial resistance to traditional control methods
- monopoly and dependence of developing countries on company who are seeking to control seed supply of world
