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
What are SNPs
Single Nucleotide Polymorphisms
- One bp difference (a different nucleotide)
EXAMPLE
Man 1 - GCA𝐀CGTTA
Man 2 - GCA𝐆CGTTA
Man 3 - GCA𝐓TCGTTA
= All DNA/gene sections are the same except for this ONE particular site
What are STRs
Short Tandem Repeats
- Short repeated sequences of DNA
Man 1 - AGA𝐂𝐓𝐀𝐂𝐓𝐀𝐂𝐓𝐀𝐂𝐓𝐀GCT 4 𝘳𝘦𝘱𝘦𝘢𝘵𝘴
Man 2 - AGA𝐂𝐓𝐀𝐂𝐓𝐀𝐂𝐓𝐀𝐂𝐓𝐀𝐂𝐓𝐀GCT 5 𝘳𝘦𝘱𝘦𝘢𝘵𝘴
a) What is a Vector?
b) What vector is used in LCA10 blindness gene therapy AND why?
a) A DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell/organism
b) A viral vector - due to its natural ability to get into cells
✰ DNA transformation ✰
Outline the 4 steps of DNA transformation
- 𝐈𝐬𝐨𝐥𝐚𝐭𝐢𝐨𝐧 of the gene & vector
- 𝐃𝐢𝐠𝐞𝐬𝐭𝐢𝐨𝐧 of gene & vector (w/ restriction enzymes)
- 𝐋𝐢𝐠𝐚𝐭𝐢𝐨𝐧 of gene & vector (w/ DNA ligase)
- 𝐒𝐞𝐥𝐞𝐜𝐭𝐢𝐨𝐧 𝐚𝐧𝐝 𝐄𝐱𝐩𝐫𝐞𝐬𝐬𝐢𝐨𝐧 of the transgenic construct
Selection = just get the DNA that has been recombined (e.g. recombinant plasmids)
✰ DNA transformation ✰
Why does the antibiotic resistance gene play a crucial role in the 4th step of DNA transformation: 𝐒𝐞𝐥𝐞𝐜𝐭𝐢𝐨𝐧 𝐚𝐧𝐝 𝐄𝐱𝐩𝐫𝐞𝐬𝐬𝐢𝐨𝐧?
With the antibiotic resistance gene, when all the DNA (e.g. all the bacterial cells) are placed in an antibiotic enviro. (e.g. a plate) - only the transformed DNA will survive and grow/glow etc.
What is Leber Congenital Amaurosis (LCA) Blindness?
A disease caused by a single nucleotide mutation in a photoreceptor gene - leading to serious vision loss or blindness within 1st few months of life
**Most common cause of inherited childhood blindness
a) What gene is affected in LCA Blindness?
b) What is this gene responsible for in humans?
a) LCA10 gene
b) A protein required for photoreceptor cells which receive light to the eye
How does the malfunctioning of the LCA10 gene impact people with LCA Blindness?
a) Faulty gene results in a short dysfunctional protein = alters the structure + function of the photoreceptor cells = photoreceptor cells not able to communicate info about light to the
brain
Why is the eye an ideal organ for developing gene therapies for LCA Blindness?
Less immune reactivity than most tissues = making a dangerous immune reaction less likely
The eye is relatively contained = the CRISPR components aren’t likely to travel to other parts of the body = lower risk of unwanted genome editing or immune responses in other tissues
What are some of the challenges faced in developing gene therapy using genome editing techniques?
Viral vectors or genome editing components will provoke dangerous immune reactions in patients
Finding ways to stop the CRISPR enzymes from
sticking around for too long - greater chance of making unwanted cuts in the DNA
Outline the 3 steps involved in curing LCA Blindness using CRISPR gene therapy
- Using an injection, the needle introduces the viral vector containing CRISPR into the eye
- The sgRNA locates and binds to the faulty copy of the LCA10 gene. CRISPR-Cas9 unwinds and cuts the DNA
- The LCA10 gene is repaired at the cut site with the correct version/gene sequence
What are the 3 Ethical approaches?
- Consequences-based approach
𝐖𝐡𝐢𝐜𝐡 𝐚𝐜𝐭𝐢𝐨𝐧 𝐩𝐫𝐨𝐝𝐮𝐜𝐞𝐬 𝐭𝐡𝐞 𝐦𝐨𝐬𝐭 𝐠𝐨𝐨𝐝 𝐚𝐧𝐝 𝐥𝐞𝐚𝐬𝐭 𝐡𝐚𝐫𝐦? (What are the outcomes - are they right or wrong) - Duty and/or Rules-based approach
𝐖𝐡𝐢𝐜𝐡 𝐚𝐜𝐭𝐢𝐨𝐧 𝐢𝐬 𝐦𝐨𝐫𝐚𝐥𝐥𝐲 𝐫𝐢𝐠𝐡𝐭 (what is the right thing to do) 𝐫𝐞𝐠𝐚𝐫𝐝𝐥𝐞𝐬𝐬 𝐨𝐟 𝐭𝐡𝐞 𝐜𝐨𝐧𝐬𝐞𝐪𝐮𝐞𝐧𝐜𝐞𝐬? - Virtues-based approach
𝐖𝐡𝐢𝐜𝐡 𝐚𝐜𝐭𝐢𝐨𝐧 𝐰𝐨𝐮𝐥𝐝 𝐚 𝐠𝐨𝐨𝐝 𝐩𝐞𝐫𝐬𝐨𝐧 𝐜𝐡𝐨𝐨𝐬𝐞?
𝐓𝐇𝐈𝐍𝐊 𝐦𝐨𝐫𝐚𝐥𝐬
What are the 5 Ethical concepts?
- Integrity
𝐃𝐨𝐢𝐧𝐠 𝐭𝐡𝐞 𝐫𝐢𝐠𝐡𝐭 𝐭𝐡𝐢𝐧𝐠 𝐞𝐯𝐞𝐧 𝐰𝐡𝐞𝐧 𝐧𝐨 𝐨𝐧𝐞 𝐢𝐬 𝐥𝐨𝐨𝐤𝐢𝐧𝐠 - Justice
𝐏𝐞𝐨𝐩𝐥𝐞 𝐠𝐞𝐭 𝐰𝐡𝐚𝐭 𝐭𝐡𝐞𝐲 𝐝𝐞𝐬𝐞𝐫𝐯𝐞 - Beneficence
𝐃𝐨𝐢𝐧𝐠 𝐠𝐨𝐨𝐝 𝐭𝐨 𝐨𝐭𝐡𝐞𝐫𝐬 - Non-maleficence
𝐃𝐨𝐢𝐧𝐠 𝐧𝐨 𝐡𝐚𝐫𝐦 - Respect
𝐇𝐨𝐧𝐨𝐮𝐫𝐢𝐧𝐠 𝐬𝐨𝐦𝐞𝐨𝐧𝐞𝐬 𝐟𝐞𝐞𝐥𝐢𝐧𝐠𝐬, 𝐧𝐞𝐞𝐝𝐬 𝐨𝐫 𝐝𝐞𝐜𝐢𝐬𝐢𝐨𝐧𝐬
