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 different about ddRAD?
