DNA manipulation Flashcards
restriction endonuclease
endonucleases that target a specific recognition site (restriction site)
DNA ligase
enzymes that anneals 2 fragments of DNA.
- form phosphodiester bonds
DNA polymerase
add nucleotides to DNA
- require a primer
taq polymerase
a heat resistant polymerase required to bind new nucleotides to single stranded DNA
-ideal temp: 72 degrees celcius
blunt ends
(restriction enzymes create) a straight cut through DNA with no overhanging nucleotides
sticky ends
(restriction enzymes create) a staggered cut through DNA which results in overhanging nucleotides
CRISPR in bacteria
- primative adaptive immune system
- defence mechanism
- used to protect against bactertiophage infection by cutting viral DNA preventing them from replicating
PAM (protospacer adjacent motif)
short sequence of nucleotides that Cas1 and Cas2 recognise.
they are signalled to extract the protospacer by cutting viral DNA just before the PAM.
Cas1 & Cas2
run along viral DNA looking for a PAM sequence. cut out a short section of DNA called the protospacer, just before the PAM sequence.
(CRISPR associated enzymes)
protospacer
section of viral DNA cut out by Cas1 and Cas2 before being inserted into the CRISPR gene
spacer
short sequences of viral DNA that are inserted into the CRISPR sequence
repeat
palindromic repeats of DNA sequences in between spacers in the CRISPR array
CRISPR array
contains repeats and spacers. part of the CRISPR array is (spacer and part of repeat) is turned into gRNA
gRNA (guide RNA)
CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it.
specific sequence determined by CRISPR to guide Cas9 to a specific site.
sgRNA (single guide RNA)
guide RNA created by scientists.
to instruct Cas9 to cut a specific site (when using CRISPR-Cas9 in gene editing)
Cas9
endonuclease that creates a blunt cut at a site specified by gRNA
CRISPR-Cas9
a complex formed between gRNA and Cas9 which can cut a target sequence of DNA.
bacteria defence mechanism against viruses
used fro genome editing by scientists
why is DNA altered after Cas9
when the DNA is cut, enzymes will try to repair it.
repair mechanisms are prone to errors so Cas9 will continue to cut that target DNA until a mutation occurs. the gene is now non-functional.
gene knock in
to add DNA segments.
scientists add them to the cell in hope they will be used in the post-cut repair process.
gene knock out
scientists prevent the expression of a target gene to understand its funtion in an organism
PCR (polymerase chain reaction)
used to amplify DNA by making identical copies
purpose of PCR
used whenever there I’d an insufficient DNA sample for testing
- analysing gene fragments for genetic testing
- paternity testing
- forensic testing
materials required for PCR
- DNA sample
- taq polymerase
- source of nucleotides
- DNA primers
(all placed in a thermocycler)
PCR stage 1
Denaturation:
- DNA sample is heated to 90-95 degrees celcius
-hydrogen bonds break to form single stranded DNA
PCR stage 2
annealing:
- single stranded DNA is cooled to 50-55 degrees celcius
- allows primers to bind
PCR stage 3
elongation:
- DNA is heated to exactly 72 degrees celcius, optimal temp. of taq polymerase
- taq polymerase binds to the primers and synthesises new strand of DNA
(cycle is repeated 35-45 times)
purpose of primers in PCR
forward primer: binds to the start codon at the 3’ end of the template strand
reverse primer: binds to the stop codon on the 3’ end of the coding strand
Allows taq polymerase to synthesise new DNA strand.
applications of gel electrophoresis
used to measure the size of DNA fragments.
- can be used to detect genetic disorders like cystic fibrosis
- can be used for DNA profiling by comparing STRs
factors affecting how a DNA fragment will run
- voltage
- gel composition
- buffer concentration
- time
STRs (short tandem repeats)
small sections of repeated nucleotides that vary in length between people
gel electrophoresis steps
- DNA samples are placed in wells at one end of the gel
- an electric current is passed through the gel
- smaller fragments of DNA move faster/travel further through the gel
- DNA is stained to make bands visible
why is a standard ladder used when reading gels
standard ladder contains DNA of a known length.
it is compared to the DNA samples being analysed to estimate their length
plasmid
small circular loop of DNA separate from the chromosome.
typically found in bacteria.
recombinant plasmid
circular DNA that is ligated to incorporate a gene of interest
bacterial tranformation
the process by which bacteria take up foreign DNA from their environment.
used to introduce recombinant plasmids into bacteria
reporter gene
gene with an easily indentifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest
electroporation
delivering an electric shock to bacterial membranes to increase membrane permeability, and increase the likelihood of bacterial transformation
heat shock
bacteria and plasmids are placed in a calcium ion solution on ice. it is then rapidly heated and cooled to increase membrane permeability, and increase the likelihood of bacteria transformation
lacZ enzyme
- lacZ gets inserted into the recombinant plasmid, already containing the insulin subunit gene.
- lacZ produces beta-galactosidase
beta-galactosidase
- creates a fusion protein with the insulin subunit protein which protects it from being digested by the cell.
- identifiable marker because it turns x-gal blue when plated with it. signals it has taken up the recombinant plasmid
how insulin is isolated from bacteria
- cyanogen bromide solution is added to bacteria
- breaks down methionine at the start of the insulin gene, separating it from the beta galactosidase
- can now be isolated and purified
genetic modification
the manipulation of an organisms genetic material using biotechnology
cisgenesis
process of inserting DNA from the same species
transgenesis
process of inserting DNA from another species
how a transgenic plant is made
- desired trait is identified and isolated
- gene of interest is inserted into the plant cell
- transformed plant cell is grown repeatedly in a lab before being transferred to fields
