UNIT 3 ➜ SAC 1 Flashcards (Gene tech, LCA Blindness, Ethical approaches)
Gene technology, LCA Blindness, Ethical approaches
What is the difference between GMOs and TGOs?
GMOs - Organisms that have had their DNA altered using genetic engineering techniques
TGOs - Organisms that have been specifically altered with foreign DNA from another species
**GMOs = Broad, any modifications
TGOs = Specific, foreign genes
What are the 4 ways GMOs can be created?
◦ Alter a gene’s expression (Express at a higher
level, in a different way etc.)
◦ Inserting a gene (Knock-in organism)
◦ Removing a gene (Knock-out organism)
◦ Inserting a foreign gene (TGO)
✰ DNA recombination ✰
a) Define DNA recombination
b) What are 2 key components used in rDNA?
a) Creating new sections of DNA sequences by combining the genetic material from 2 or more different sources
b) Restriction enzymes & DNA ligase
✰ DNA recombination ✰
How is DNA recombination possible?
(HINT - Universal)
DNA of every organism is made up of the same building blocks (nucleotides) = allowing the gene of one organism to be moved + expressed in another organism
✰ DNA recombination ✰
a) What is the role of Restriction enzymes?
b) What are the 2 ways that DNA can be cut using these enzymes?
a) A group of enzymes that cut DNA molecules at specific recognition sites
b)
◦ Sticky ends (Single stranded overhangs)
◦ Blunt ends (Cut symmetrically, no overhangs)
✰ DNA recombination ✰
a) What is the role of DNA Ligase?
b) What is important in this process before 2 DNA fragments can be joined together?
a) An enzyme that joins two DNA fragments together (through reforming the strand’s phosphodiester bonds)
b) That the 2 DNA fragments have been cut by the same restriction enzyme = same ends (sticky or blunt)
✰ CRISPR-Cas9 ✰
a) What does CRISPR stand for?
b) What is CRISPR?
a) Clustered Regularly Interspaced Short Palindromic Repeats
b) An endonuclease complex that can target specific stretches of DNA + edit it at very precise locations
✰ CRISPR-Cas9 ✰
a) Where does CRISPR come from?
b) What are the 2 key components needed for CRISPR to work?
a) Comes from bacteria - used as immune system protection by editing the DNA of invading viruses
b) The sgRNA guide & Cas9 enzyme
✰ CRISPR-Cas9 ✰
a) What is the role of the sgRNA guide?
b) What is the role of the Cas9 enzyme?
a) Locates and binds to the target piece of DNA
b) Unwinds and cuts the DNA
✰ CRISPR-Cas9 ✰
What component of CRISPR stops Cas9 from cutting undesired sections of DNA?
Protospacer Adjacent Motif (PAM)
PAM is a short nucleotide sequence within the viral genome
CRISPR has spacers that contain past infections (allow CRISPR to easily combat the virus if it attacks again)
◦ If PAM is present, Cas9 knows to cut the DNA
◦ Spacers containing past infections don’t contain a PAM = Cas9 isn’t unable to cut the spacers
✰ PCR ✰
a) What does PCR stand for?
b) What is the purpose of PCR?
a) Polymerase Chain Reaction
b) Amplifying target sequences of DNA by producing many copies of a specific segment
✰ PCR ✰
Outline the 3 steps of PCR (including the required temperatures of each stage)
𝟏. 𝐃𝐞𝐧𝐚𝐭𝐮𝐫𝐚𝐭𝐢𝐨𝐧
Heating the target DNA stands to 95°C - breaking the hydrogen bonds and separating the DNA into 2 singular strands
𝟐. 𝐀𝐧𝐧𝐞𝐚𝐥𝐢𝐧𝐠
The temp cools down to 55-65°C - hot enough that the strands remain separated but cool enough that primers can attach/bind to the 3 prime ends of each strand
𝟑. 𝐄𝐱𝐭𝐞𝐧𝐬𝐢𝐨𝐧
72°C - Taq polymerase (type of DNA polymerase) extends the primers by adding nucleotides to the 3 prime ends, making a complementary strand - and therefore double-stranded DNA
✰ PCR ✰
a) What components go in the Reaction mix (before PCR is started)?
b) How long is each cycle AND how many cycles are completed - usually?
a) DNA sample (target sequence), Primers, Nucleotides, Taq polymerase, Buffer solution (provides the right conditions)
b)
A PCR cycle = goes for approx. 20 mins
Undergoes 35 cycles: produces 68 billion copies
✰ Gel Electrophoresis ✰
Define Gel electrophoresis (what is it?)
A process that uses gel to separate large molecules based on size, charge and shape
✰ Gel Electrophoresis ✰
Outline the process - what steps are involved?
**Focus on using DNA samples
After the gel is prepared and the samples are loaded into the wells near the negative terminal (due to DNA negative charge):
◦ An electric current is applied, allowing the molecules to move through the gel (towards the positive terminal for DNA)
◦ Molecules of different sizes become spread out on gel’s surface (smaller molecules travel further, large molecules travel less due to size)
◦ Molecules can be visualised as bands + DNA can be strained and visualised under UV
**KEY takeaways
Prepared gel and DNA samples ⇨ Loaded into wells near negative terminal ⇨ Molecules travel (small = farther, larger = less) ⇨ DNA fragments visualised as bands
