DNA Manipulation Flashcards
Describe the denaturing phase of polymerase chain reaction
Denaturing - Heat DNA to 90oC to separate the DNA into single strands by breaking hydrogen bonds between complementary bases.
Describe the primer annealing phase of the polymerase chain reaction
DNA Primer Annealing - Cool to approximately 50oC to attach/anneal DNA primers to the single-stranded DNA.
Describe the extension phase of the polymerase chain reaction
Extension- Heated to approximately 72oC - DNA Taq polymerase copies the single DNA strands in a 3’ to 5’ direction using complementary base pairing.
State how many times the polymerase chain reaction is completed
35+ times
Name the fours stages of PCR
Denature
Primers Anneal
Extension
Repeat 35+
State the three temperatures of PCR
90 C
50 C
72 C
Outline the four stages of PCR
Denaturing - Heat DNA to 90oC to separate the DNA into single strands by breaking hydrogen bonds between complementary bases.
DNA Primer annealing - Cool to approximately 50oC to attach/anneal DNA primers to the single-stranded DNA.
Extension- Heated to approximately 72oC - DNA Taq polymerase copies the single DNA strands in a 3’ to 5’ direction using complementary base pairing..
Repeats - Process is repeated 35 times.
Reverse transcriptase is an
enzyme that copies mRNA into copy DNA
DNA primers are
Short single-stranded DNA fragments that attach to DNA to allow the binding of DNA taq polymerase
State the funciton of an endonuclease
Cut DNA at a specific recognition site
Describe how to create a DNA profile
Cut the DNA sample with an endonuclease
Place DNA sample in a well at the negative end of a gel
Turn on the electricity and the DNA fragments will move towards the positive end.
They will seperate based on size and charge.
Compare with standards which are of know size, that were also placed in the first well, to estimate the size of the other fragments.
State the purpose of standards in an agarose gel
These are DNA fragments of known size (bp) that can be used to compare and then estimate the size of the other fragments.
An endonuclease is
Bacterial enzyme that cuts DNA at a specific recognition sequence
Gel electrophoresis sorts DNA based on
size and charge
Gene cloning is
Making multiple copies of a gene, usually within bacteria in order to express the gene product
A plasmid is a
Small ring of bacterial DNA can be used as a vector for DNA recombination and insertion
Bacterial transformation is a process by which
a bacterial cell takes up a recombinant plasmid and expresses the genes of the plasmid
Reverse transcriptase is a
(Retrovirus) enzyme that copies mRNA (e.g. human insulin mRNA) into c.DNA so that it contains no introns.
Define a GMO
An organism whose genome has been altered
Define a TGO
Genetically modified organisms where genes from a different species are added to their genome
In gel electrophoresis DNA moves from the ______ end to the ______ end.
Negative to positive end
A DNA probe is
a single-strand segment of DNA which is (radioactively) labelled.
Outline the steps taken to produce human insulin from bacteria.
Isolate gene for insulin chain A
Use reverse transcriptase to get c.DNA from isolated mRNA so that it contains no introns.
Cut gene and plasmid using the same endonuclease.
Stick insulin gene into plasmid using DNA ligase which joins the sugar phosphate backbone.
Transform bacteria using heat or electroporation and check for successful transformation by growing on an antibiotic plate with ampicillin and then an antibiotic plate with tetracycline
Bacteria with no plasmid will not grow on the ampicillin plate, successfully transformed bacteria with the non-recombinant plasmid will grow on both plates and the successfully transformed bacteria with the recombinant plasmid will only grow on the ampicillin plate.
Purify plasmids, cut with an endonuclease and stick β-galactosidase gene into plasmid using DNA ligase
Transform bacteria using heat or electroporation and check for successful transformation by growing on X-gal agar plate
Bacteria with no β-galactosidase gene will appear white and bacteria with the β-galactosidase gene will appear blue, collect blue colonies
Repeat for insulin chain B
Purify insulin chains A and B and join them together to create a functional insulin
CRISPR Cas 9 was originally discovered in ..
Bacteria
Describe the role of CRISPR in bacteria.
Primitive adaptive immune system this means that
Invading viral DNA is cut and stored as spacers in the CRISPR array providing a memory of viral infections
If the same virus reinfects the bacteria, gRNA is transcribed and attached to a Cas9 endonuclease forming a gRNA-Cas9 complex
gRNA guides Cas9 to the viral DNA and Cas9 recognises the PAM sequence and then cuts the viral DNA, preventing destruction of the bacteria.
What are the two components of using CRISPR technology in other organisms?
Cas 9 enzyme and a single guide RNA ( sgRNA)
State enzymes used in gel electrophoresis
Endonuclease
State enzyme used in PCR
DNA Taq polymerase
Describe the steps in using CRISPR technology to edit genomes in other organisms
Identify the nucleotide sequence of the ______ target gene.
Make single guide RNA which is complementary to the ____ target DNA.
This is joined with Cas9 to form the sg.RNA Cas9 complex.
Sg.RNA guides Cas9 to the _____ gene and Cas9 cuts the DNA.
Transcription/no transcription of the _____ gene will occur and a functional ____ protein/non-functional _____protein is produced.
State what viral DNA is stored as in bacteria as part of CRISPR.
Spacers
Describe the function of PAM in a prokaryote
Acts as the binding site for the cas9 endonuclease and allows cas9 to cut the DNA sequence.
The PAM sequence plays an essential role in distinguishing self (CRISPR) from non-self (viral DNA).
Allows for faster recognition of viral DNA when it enters a cell
Use the explain scaffold to justify an organism as being a genetically modified organism.
Because a genetically modified organism is one whose genome has been altered whereas a transgenic organism has had a gene from a different species added to its genome
Then as the ____ has had its genome altered without the addition of a different species gene
It is therefore a genetically modified organism
Use the explain scaffold to justify an organism as being a genetically modified organism and transgenic organism.
Because a genetically modified organism is one whose genome has been altered whereas a transgenic organism has had a gene from a different species added to its genome
Then as the ____ has had its genome altered with the addition of a different species (_______) gene
It is therefore a genetically modified organism and transgenic organism.
Purpose of PCR
To amplify target DNA, creating multiple identical copies this means that the volume of DNA is increased so there is enough to be analysed using gel electrophoresis
