CRISPR AND DNA MANIPULATION Flashcards
SAC 2
Requirement for polymerase chain reaction(PCR)
1)Taq polymerase
2)dNTPs
3)primers
4)Dna to be amplified
5)Buffer(Control pH)
What happen to DNA after each PCR cycle
The copies of DNA double every cycle (2,4,8,16..)
Use for PCR
forensic,parentage testing,diagnoisis of hereditary diseases,gene cloning
why plasmids are advantegous when being used to transfer DNA from organism to another
Able to be copied many times within bacterial cells; circular – much more stable than linear fragments;
small enough to be able to be distinguished from the main bacterial chromosome yet large enough to
be extracted and manipulated; easily engineered to carry a number of different genes.
Define restriction enzyme
Restriction enzymes are used to cleave or cut the DNA at a specific restriction site to produce sticky or
blunt ends through breaking the phosphodiester bonds in both strands of the DNA.
define ligase
Ligase is the enzyme responsible for joining a gene of interest into the plasmid through the catalysing
of phosphodiester bonds.
Why are sticky ends prefered over blunt ends
Sticky ends are preferred over blunt ends as it allows for the overhang of nucleotides on the plasmid
vector where the gene of interest with complementary nucleotides can efficiently join.This was they are less prone to misalignment, and making ure only gene of interest is added.
What is bacterial transformation
Bacterial transformation is a process where bacteria takes up genetic material (transformed or non
transformed plasmids) from its surrounding environment through the cell wall.
name all ethical concepts
Respect,integrity,justice,Beficene,non-maleficence,respect(6)
define intergrity
an ethical concept that
encourages a full commitment to
knowledge and understanding as
well as the honest reporting of all
sources of information and results
define justice
an ethical concept that
encourages fair consideration of
competing claims, and ensures
that there is no unfair burden on a
particular group from an action(marginalised by action).Justice prioritises the fair distribution of resources,
as well as equal access to the benefits of an action, policy, investigation,
or research.
define non–maleficence
– the commitment to minimising harm.or when harm is
unavoidable, ensuring that the
harm is not disproportionate to
the benefits from any position or
course of action
define respect
an ethical concept that
encourages the acknowledgment
of the intrinsic value of living
things, and considers the welfare,
beliefs, customs, and cultural
heritage of both the individual and
the collective
During the polymerase chain reaction, explain why the temperature is reduced in the annealing stage and then increased in the elongation stage
he temperature must be reduced in the annealing stage to allow for hydrogen bonds to form between the primer and the complementary region of the single strand of DNA.1The temperature is then increased to 72 °C during the elongation stage as this is the optimal temperature for polymerase to extend the primer and create a new strand of DNA.
insulin production summary
1.a vector plasmid is chosen with ampR and tetR with known restriction enzyme sites(BamH and ECORI)
2.Insulin A and B subunit genes(wihout introns) cut and ligate to form recombinant plasmid.
3.Recombinat plasmids are then added to bacteria in seperate container
4-Bacteria gorwn on amp plate to see if any plasmid were taken up then further some colonies are grown on tet plate to see if recombinat plasmid were taken.
5-lac z gene is inserted into plasmid,usinf EcoRI which produces ß-galactosidase,to make fusion protein with insulin gene.
6-Plasmids containing lacZ added to bacteria
to create transformed bacteria
7-The ß-galactosidase
enzyme was known to convert a compound called X-gal, he E. coli were plated
on agar plates containing ampicillin and X-gal. Colonies that grew and were blue in
colour were identified as containing recombinant plasmids due to the presence of
ß-galactosidase
8-Insulin subunit genes expressed attached
to large -galactosidase proteins.no stop codon after ß-galactosidase and a methionine at start of insulin chain,which is then broken by cyanogen bromide.
9-botj chains mixed together and make disulphide bonds
Steps involved in CRISPR gene editing
1.syntjetic sgRNA is created in the lab with complementary sequence to the gene of interest.
2.Cas9 and sgRNA combined in a mixture to creat cas9 complex
3.sgrna and cas9 mixture added injected to a cell
4.cas9 find the pam sequence and checks wheather the sgrna align with the DNA
5.Cas 9 cut the seleccted sequence of DNA
6.When repairing the cell,sceintist may try to inject template of og the gene of interest and hope it will liage in the gap
steps involved in CRISPR-Cas9 defence system in the bacteria
Exposure-the bacteriaphage injects DNA into a bacterium,which identifies as foreign substance.Cas 1 and Cas2 identify PAM sequence and a cut a short peice of viral DNA,which is then inserted into the CRISPR array
Expression-the CRISPR spacers are transcribed along with half a palindrome
from the repeat either side of it, and converted into an RNA molecule known as
guide RNA (gRNA). gRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA forms a hairpin loop-like structure from the transcribed
palindromic repeats either side of the space.
Termination– The CRISPR-Cas9 complex then scans the cell for invading
bacteriophage DNA that is complementary to the ‘mugshot’ on the gRNA. When it
does, Cas9 cleaves the phosphate-sugar backbone to inactivate the virus. Cas9 contains
two active sites to cut both strands of DNA and create blunt ends
Explain gene knock out
a technique in
gene editing where scientists
prevent the expression of a target
gene to usually understand its function in
an organism.When a break occurs in DNA,a cell will try to fix it.If there no other copies of DNA ,othe cell will try to repair itself with (non-homologous end joining),this will cause some sort of mutation in the gene which makes it unfunctional.
Describe the PCR process
denaturing-The temperature is brought is to 95 degress to denature the DNA strand and break the hyndrogen bond between the two strands to seperate them.
annealing-the temperature is then brought to 55 for the DNA primers to bind to complementary base.
Elongationg-the temperature is then brough to 72,which is the optimal temperature for the taq polymerase enzyme,which exterd the primers by adding more bases.
repeats-the steps 1-3 are repeated to creat more copies, as the DNA segments doubles every cycle.
role of PAM sequence
1.cas 1 and cas 2 work in tandom.when they come across any PAM sequence,they are signaled to extract a protospacer from infecting viral.
Cas 9 only looks for PAM sequence then checks if the sgrna is complementary to the gene of interest.
Why doesnt the bacteria cleave its own crispr array.
Because cas-9 first looks for any PAM sequecne, the CRISPR array itself doesnt have any PAM sequence as Cas 1 and Cas 2 cut just before the PAM,not including it.
How the CRISPR-Cas9 defence system works-describe exposure
Exposure-bacteriaphage injects it DNA into the bacterium,which is identified as forenign subsbstance as Cas 1 and Cas 2 look for PAM sequence and cut out a short sequence of the viral DNA and inserts in the the bacertia’s CRISPR array.
How the CRISPR-Cas9 defence system works-describe Expression
the CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it, and converted into an RNA molecule known as
guide RNA (gRNA).gRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA.gRNA forms a hairpin loop-like structure from the transcribed
palindromic repeats either side of the spacer.
blunt end
the result of a straight
cut across the double-stranded
DNA by an endonuclease resulting
in no overhanging nucleotides
How the CRISPR-Cas9 defence system works-Extermination
The CRISPR-Cas9 complex then scans the cell for invading
bacteriophage DNA looking for a PAM sequence and seeing if the mugshot (sgrna) is complementary to it.
Cas9 contains
two active sites to cut both strands of DNA and create blunt ends
guide RNA (gRNA)
RNA which has a specific
sequence determined by CRISPR
to guide Cas9 to a specific site
band
a line seen in the gel after
running gel electrophoresis that
corresponds to a collection of DNA
fragments of a specific size
ethidium bromide
a fluorescent
dye that binds to DNA fragments in
a gel and allows them to be easily
visualised under ultraviolet light
electrode
conductors of electricity
that are attached to both ends of a
gel allowing an electrical current to
pass through it
agarose gel
a sponge-like gel used
in gel electrophoresis that contains
pores for DNA fragments to move
through
standard ladder
a mixture of DNA
fragments of known length that are
used to infer the size of fragments
in a sample
buffer
an ion-rich solution that
carries electrical current through
the agarose gel
factor that effect the speed of the movement of DNA fragments in gel electroporesis
strenght of electric feild-increases volatage.
size of the DNA fragments
concentration of the buffer
decribe the process of making transgenic plants
genetic identificcation-GOI is and identified and isolated which contains a genes of a useful trait from another organism.
Gene delivery-GOI is then inserted into cells directly inserted or through bacterial plasmid.
Gene expression-the transformed cell is then grown repeatedly (regenerated) using
plant tissue cultures under sterile conditions before being applied in the field foragricultural use. The GM host organism is now able to express the new transgene as a useful protein and can regenerate itself
consequence based approach
maximise positive outcomes,while minimising the negative.individuals driven by consequence
duty based aproach
aim is to comply with all rules regulations and responsibilites,with less regard to the consequence.
virtue based approach
driven by moral belief of a person,often argues action can be justifies simply because it is charitable,or caring or good
