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
