Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
16 Cards in this Set
- Front
- Back
|
What are the two major methods for DNA sequencing? |
Sanger Coulson (chain termination) Maxam Gilbert (chemical degradation) |
|
|
Components required for SC sequencing? |
DNA Pol NT analogues ssDNA Labelling (historically RA, now fluoro) |
|
|
What is the difference between a dNTP and ddNTP? |
dNTP has OH on C3 and H on C2, ddNTP has H on C3 and C2 |
|
|
Why is SC sequencing also known as chain termination sequencing? |
Use of ddNTPs randomly means phosphodiester bonds cannot form at those points, so strand synthesis is terminated and a fragment is produced |
|
|
What radiolabel is classically used in SC sequencing? |
35S-dATP |
|
|
What technique is used to analyse the products of SC sequencing? |
PAGE |
|
|
PAGE properties for SC sequencing analysis? |
10% concentration, 40cm+ length 0.2-0.6 thickness, denaturing (50deg, contains urea) high voltage (~1700volts) |
|
|
Fluoro dyes used in bioscience are selected for depending on which properties |
ExMax (excitation wavelength) EmMax (emission wavelength) |
|
|
Which radiolabel has been classically used in MG sequencing? |
32P-dATP |
|
|
MG sequencing reactions produce fragments ending in what? |
G alone C alone G or A C or T |
|
|
Describe the MG cleavage reactions for the purines. |
G+A methylated by dimethyl sulphate Heat+ pH7 - removes methylated G Heat + dilute acid - removes both methylated purines |
|
|
Describe the MG cleavage reactions for the pyrimidines |
Hydrazine splits C or T 2M NaCl protects T, so hydrazine splits C only |
|
|
Once MG cleavage reactions have taken place, what reaction is used to separate fragments? |
Piperidine cleaves DNA backbones at all modified bases |
|
|
What are the newest potential sequencing methods to be used? |
Pyrosequencing MALDI-TOF-MS |
|
|
Typical NT structure includes what? |
Phosphate group Deoxyribose sugar Nitrogenous base |
|
|
Stages of end-strand labelling in MG sequencing? |
RE acts at ends of DS fragment Polynucleotide kinase allows 32PdATP label Heat+DMSO breaks H bonds - separation Products on PAGE - purify to select 1 strand |
