Biology Unit 3 SAC 1 Flashcards
Restriction Endonuclease
Type of enzyme that acts like the molecular scissors, cutting nucleic acids strands at recognition sites (AKA Restriction enzymes)
Endonuclease
Type of enzyme which breaks phosphodiester bonds between two nucleotides in PPC
Ligase
Enzyme that joins two molecules together by catalysing formation of phosphodiester bonds
Polymerase
Enzyme that synthesis polymer from monomer such as DNA strand from nucleic acids.
Primer
Short single strand of nucleic acid that acts as starting point for polymerase enzyme to attach on
Explain the 3 steps of CRISPR-Cas9 in bacteria
1) Exposure - The bacteriophage injects its viral DNA into the bacterium which is recognized as foriegn. Cas1 and Cas2 recognizes the PAM sequence which signals to extract protospacer from viral DNA enzymes work together to cut a section of the viral DNA just before the PAM and introduces it to the bacteriums CRISPR gene becoming a spacer.
2) Expression - The CRISPR spacers are transcribed and converted into RNA molecules known as gRNA. the gRNA binds to the cas9 to create CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA
3) Extermination - CRISPR-Cas9 complex brouses cell for other invading bacteriophage complementary to gRNA sequence and when it does the Cas9 inactivates the virus through the phosphate-sugar backbone. Cas9 contains 2 active sites which cuts both strands of DNA and creates blunt ends.
Explain the 8 steps of CRISPR-Cas9 in gene editing
1) Synthetic sgRNA is created in lab which has complementary spacer to target DNA scientists wish to cut/silence
2) Cas9 enzyme obtained with appropriate PAM target sequence
3) sgRNAand Cas9 are put in a mixture together to bind creating CRISPR-Cas9 complex
4) sgRNA and Cas9 mixture is injected into specific cell such as Zygote
5) Cas9 finds PAM target sequence and check if sgRNA aligns with DNA
6) Cas9 cuts selected sequence of DNA
7) The DNA has blunt end which the cell will try to repair
8) When repairing the DNA the cell may introduce new nucleicotides into DNA, and scientists may inject particular nucleotide in hopes it will ligate the gap
Applications of CRISPR-Cas9 in RESEARCH
- Attaching fluorescent protein to Cas9 to locate specific gene in genome.
-Disrupting the expression of genes to see the effect of that protein being knocked out. This helps scientists identify the function of specific genes.
What makes CRISPR unique as restriction endonuclease in a tool for human research
CRISPR is unique as an endonuclease because it can be ‘programmed’ with different g/sgRNA molecules to cut at different sequences, unlike other endonucleases which can only cut at one specific recognition site
(e.g. the restriction endonuclease SmaI only cuts at CCCGGG, whereas CRISPR can be programmed to cut at any desired sequence by changing the gRNA/sgRNA molecule).
Why CRISPR-Cas9 should be inserted into a zygote to produce a change for whole oragnsim
To produce changes throughout the whole oragnsim it firstly starts of with the zygote the first cell of a new oragnsim because the zygotes DNA is passed down to all subsequent cells, as we are too developed now.
What is the overall structure of DNA and RNA with reference to the bonds
DNA is double stranded helix, each strand with a backbone of sugar-phosphate linked by covalent bonds called phosphodiester bonds. The complementary base pairing (A, T, C, G) are held by hydrogen bonds.
RNA is single stranded and instead of a Thymine base it contains ucrail instead.
Name and define 5 bioethical concepts
1) beneficence - the commitentment to maximising benefits, and minimsing risks
2) integrity - the commitment to searching knowledge and understanding. the honest reporting of all sources
3) respect - the acknowledgement of intrinsic values of all living things, and the
4) justice -
5) non-malefience - non harm
name and define the 3 bioethical approaches
1) consequences based - the aim to maximise positive outcomes and miniside negative outcomes
2) duty-rule based - Promotes responsiblity of the agent and importance of duty
3) virtues based - Emphaises indivduals goodness and promotes acting in accordance with values of moral person
What is the purpose of PCR?
The purpose of PCR is to amplify a specfic segment of DNA by making multiple identical copies, used by scientists when there is insufficient amount of DNA sample.
- Forenstic testing
- Paternity testing
- Analysing gene fragments for genetic diseases
Name and explain 3 steps of PCR with temperatures
1) Denaturation - DNA is heated to 90-95 degrees to break hydrogen bonds between bases forming single stranded DNA
2) Annealing - Single stranded DNA is cooled to 50-55 degrees to allow primers to bind to complementary sequences
3) Elongation - The DNA again is heated to 72 degrees allowing Taq polymerase to work optimally binding to the primer and begins synthesisng new complementary DNA strand
4) Repeat the cycle multiple times for multiple copies.
Gel electrophoresis
Technique to separate DNA fragments based on their molecular size.
The set of gel electrophoresis:
- Wells: Indent in gel into which a DNA sample is loaded
- Standard ladder: mixture of DNA fragments of known length that are used to infer the size of fragments in a sample
- Buffer solution: Ion rich solution which carries electrical current through agarose gel.
-Porus Gel: The gel has porous (holes) in them - Electrodes: One positive and negative electrode, negative positioned near Wells and positive opposite end of gel. Since DNA is negatively charged it will be attracted to the positive electrode. When the electrical current is applied the DNA fragment will move through the pores towards positive.
How the size DNA fragments affect their movement through the gel
The smaller fragments move furthur to the positive electrode due to gel agarose having smaller pores allowing for the movement of DNA. (faster and furhur)
Larger fragments move slower due to being to large to pass through the pores of the agarose gel.
Plasmid
Small circular loop of DNA found in bacteria
Vector
Way of introducing foriegn DNA into oragnsim (plasmids being a popular vector in bacterial transformation)
Bacterial Transformation
The process where bacteria takes up foreign DNA and scientists use this process to introduce recombinant plasmids into bacteria
Recombinant plasmid
Small circular loop of DNA vector ligated to include a gene of interest
Components of plamid vector
-Antibiotic resistance gene: gene which confers antibiotic resistance
-Orgin of replication: sequence found in prokaryotes signalling start site of DNA replication
- Reporter gene: Gene with easily indentifable phenotype used to identify whether a plasmid
has taken up the gene of interest.
Explain RNA processing
The modification of pre mRNA to mRNA used in translation.
1) Alternative splicing - Removal and rearranging of exons to give rise to many different mRNA strands coding for different proteins.
2) Addition of 5-methyl gap cap 3-poly A tail - Serves to stablise mRNA molecule preventing it from degrading and allowing it to bind to ribsome during translation.
