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 elongation phase of the polymerase chain reaction
Elongation - 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
State the purpose of the polymerase chain reaction
To amplify the DNA so that there is enough to analyse
Name the fours stages of PCR
Denature/Dehydridise
Primers Anneal
Extension/Elongation
Repeat 35+
State the three temperatures of PCR
90 C
50 C
72 C
Outline the four stages of PCR
Denaturing - Heat DNA to approximately 90C to separate the DNA strands.
Primer annealing - Cool to attach approximately 50C to attach primers at the 3’ end of each DNA strand.
Extension (elongation) - Heat to 70C so Taq polymerase can copy strands in a 3’to 5’ direction.
Repeats - Process is repeated 35 times.
Reverse transcriptase is a
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 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.
State the purpose of standards in an agarose gel
These are DNA fragments of known size (bp) that can be used to 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 plasmid and expresses the genes of the plasmid
Reverse transcriptase is a
Retrovirus enzyme that copies mRNA into copy DNA
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.
How are bacteria that have been transformed identified?
Plasmid includes an antibiotic resistant gene. Bacteria are grown on agar containing that antiobiotic so that only those that are transformed will survive
Social implications may impact
person’s financial position, lifestyle and reproductive decisions or many people.
Social implications may impact
person’s financial position, lifestyle and reproductive decisions or many people.
Ethical considerations are
philosophical, moral or religious issues related to the impact of these technologies.
Biological impacts
relate to organisms used in and affected by the technology, its safety and short-term or long term changes to human biology.
Genetic screening is …
testing an individual to identify the presence of a particular allele.
Steps in gene cloning are …
Identify gene of interest using reverse transcriptase to get c.DNA
Cut gene and plasmid using the same endonuclease.
Stick gene into plasmid using DNA ligase which joins the sugar phosphate backbone.
Transform bacteria using heat or electrical shock
Check for successful transformation (usually using an antibiotic or GFP). Untransformed bacteria will die with the antibiotic. Successful will express the gene making the protein.
Steps to modify plants using agrobacteria include
Identify gene that codes for the protein of interest and cut this out using an endonuclease. Cut a plasmid. Join the gene of interest to the plasmid using DNA ligase. Transform Bacteria (Agrobacteria) with the plasmid. Bacteria will then enter the plant’s cells and then inject the plasmid DNA into the host genome. This will then be able to be expressed.
CRISPR stands for
clustered regularly interspersed short palindromic repeats.
CRISPR Cas 9 was originally discovered in ..
Bacteria
What is the role of CRISPR in bacteria?
Adaptive immune system as a memory of viral DNA that the cell has encountered before.
What are the two components of using CRISPR technology in other organisms?
Cas 9 enzyme and a single guide RNA ( sgRNA)
Describe the Steps of using CRISPR Cas 9 to edit a genome
- Synthetic gRNA is created in a lab. This means that it is complementary to the target DNA that scientists wish to cut.
- A Cas9 enzyme which recognises an appropriate target PAM sequence and the sgRNA are added together in a mixture. This means that they bind together to create the sgRNA-Cas9 complex.
- The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote. This means that it is only one cell.
- The Cas9 recognises the target PAM sequence and Cas9 cuts the selected sequence of DNA. This means that a blunt end cut will be formed that the cell will attempt to repair. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site.
Explain the process of attenuation, when there is low levels of tryptophan present.
Because tryptophan is present, the ribosome runs past the tryptophan codons and stops at the stop codon between domains 1 and 2.
This then prevents 2 from pairing with 3, leaving 3 free to pair with 4, forming a ‘hairpin’.
Therefore putting tension on the attenuator, so the mRNA pulls away from the DNA, causing RNA polymerase to fly off and ending transcription of the structural genes.•
Explain the process of attenuation, when there is no tryptophan present.
Because tryptophan is absent, the ribosome pauses at the two tryptophan codons, waiting for a tRNA carrying tryptophan.
Then Domain 1 is covered and allows Domain 2 to pair with 3, preventing Domain 3 pairing with 4.
Therefore this creates a hairpin, but because it’s a fair way from the attenuator, the mRNA does not pull away from the DNA, and the RNA polymerase continues to slide along, transcribing the structural genes of the operon (TrpE - TrpA).
