Jordans Notes 8 Flashcards
How does detergent break apart cell membranes?
attaches to lipids
What is added during the cell lysis stage of spin
column extraction?
Detergent → attach to lipids
proteinase K → denature proteins including nucleases
chaotropic salts → destabilise hydrogen bonds, hydrophobic interactions
What are the two washes that occur following the
binding stage of spin column extraction?
- small amounts of chaotropic salts to remove remaining proteins or pigments/contaminants
- high concentration ethanol to remove remaining salts
What does the 5’ end of DNA end with?
phosphate group
What are PCR primers?
single-stranded DNA fragments complimentary to sequences that flank the target region that we want to amplify (the amplicon)
What determines the size of the PCR product?
The distance between the primer binding sites
What temperature is the extension stage of PCR
done at?
72℃
A non-protein compound or metallic ion required
for enzymes role as a catalyst?
Cofactor chemicals
e.g. Mg+
What does first and second generation sequencing
have in common?
Sequencing by synthesis
Which sequencing method doesn’t sequence by
synthesis?
MinIon (3rd gen)
What length are nucleotide short tandem repeat
units usually?
2-5 base pairs long
What is genotyping?
Determining which genetic variants (alleles) an individual
possesses
How does NGS do genotyping?
by looking at single nucleotide polymorphisms (SNP)
What are the three main types of SNP genotyping?
Genotyping by sequencing (GBS)
Restriction-Site-Association DNA sequencing (RADseq)
Double-digest Restriction-site-Associated DNA sequencing
(ddRAD)
What is the start of all SNP genotyping processes?
Using a restriction enzyme digest
- splits DNA with an offset to produce sticky ends
What is it called when only a subset of the genome
is analysed?
Reduced-representation sequencing
How do you reduce genome complexity
restriction enzyme digesting
- finds specific sequences and then cuts them (”digests”)
- creates sticky ends from offset cuts
- other sequences or products can then ligate to the sticky ends
- effectively creates a smaller “sample of the entire genome”
What is different about ddRAD?
What enhances the binding of DNA to the silica
membrane/beads
ethanol and a hydrophobic environment
Added in the “binding stage” which is right after lysis
What makes DNA more stable and dissolve more
easily?
A slightly basic pH
during elution stage
How is elution success maximised? (4 ways)
Make sure all salts are washed off during Wash 2 (repeat wash 2)
Make sure no ethanol remains (centrifuge until dry)
allowing buffer to soak for a few minutes before centrifugation
repeating elution step
What two stages of spin column extraction can be
repeated to ensure maximum DNA yield?
Wash two with ethanol to remove all salts
Elution
What are the 5 stages of spin column DNA
extraction?
- Lysis (Detergent, Pro K, Chaotropic salts)
- Binding (ethanol) → salt bridge
- Wash 1 (>chaotropic salts) → proteins + pigment
- Wash 2 (<ethanol) → salts
- Elution (AE buffer of water) → rehydrates DNA
10 Steps for Ethanol precipitation extraction
- Cell lysis
- incubate with denaturation buffer
- mix isopropanol by inversion → precipitates DNA
- centrifuge to form DNA pellet
- remove isoprop supernatant
- wash pellet with ethanol
- Sample centrifuged
- remove ethanol supernatant
- air-dry pellet
- dissolve pellet in storage buffer by incubating at 65℃
What can be difficult about the ethanol
precipitation extraction method?
removing supernatants and resuspending pellets
requires experience and technique
What is the process to chelex extraction
- chelex added to sample
- Thoroughly immerse sample in chelex using vortex and a quick centrifuge
- sample incubated at 95℃ for 20 minutes (vortex halfway through)
- Centrifuged to pellet chelex resin
- supernatant containing DNA is removed
What 3 things does heating chelex sample to 100℃
achieve?
ruptures cell membranes
destroys cellular protein
denatures DNA → single stranded
What is the ultimate purpose of PCR?
to target and amplify a specific genomic region of interest
to then be sequenced
What are the 5 ingredients for PCR?
- Primers
- Taq Polymerase
- Genomic DNA (template)
- Co-factor chemicals (Mg , buffer, KCl)
- Deoxyribose nucleoside triphosphate (dNTPs)
What is a nucleoside?
A base pair without the phosphate group
