Sanger Sequencing, SDS-Page, Blotting, Sequencing Coverage

what is a dideoxynucleotide

No OH group of 3’ of sugar - just H

why does a deoxynucleotide interrput DNA replication

there is no OH group on 3’ side for polymerase to add to

what is Sanger DNA sequencing

chain termination sequence, DNA is denatured for single strands, short DNA primer added so DNA pol can start. Add ddntp (radiolabeled). Let DNA pol run. load fragments into polyacrylamide gel and read

how to read DNA sequnce of off sanger sequencing gel

from bottom to top

how has sanger sequecing been improved with advancemns in detection technology

gel is placed in a capillary tube, electrophoresis occurs through the tube. light shines through and the computer detects how long it takes for bands to fall thought, bases are designated by different colors

what are the next generation detection - faster cheaper

New Sanger (technically not considered) 0 flourcent labeled ddNTP instead of radioactive, Illumina - reversible and pyrosequencing - pyrophosphates released and detected by computer

what is the SDS Page - Gel used for

SDS denatures proteins, then run on SDS page gel to observe size

what does induced/treated mean in terms of SDS page experiments

make the cell make the protein

explain the basic process of a western blot

proteins denatured in SDS and run on gel, transferred to thin membrane and washed with antibody, the antibody sticks to desired protein and then washed away other unwanted ones. the antibodies don’t go through gel and so we blot it onto a membrane

what do first and second antibodies do

1st attaches to protein and 2nd attach to first and label protien so you can see them

why do we blot the gel onto a membrane/ paper before the washes

because antibodys wont go throught he gel so we have to blot them

southern blot is for

DNA

northern blot is for

RNA

western blot is for

protein

what are the labeled probes used in Southern northern, western blot

something that helps up test for the presence of what we are looking for, S or N - complementary of DNA or RNA, W - secondary antibody

if we can only reliably sequence small lengths of DNA, how do we sequence whole chromosomes?

use sequence assembly and overlap fragments to put together entire chromosome - with computer

how is sequence assembly done

fragmentation (read)s, sequencing with computer, reads aliened, and consensus reads happen

read

short sequence/ fragment - line on sequencing

fold coverage

lowest amount of bases lined up

consensus sequence

what computer decides is the genome (DNA sequence)

how are contigs and consensus related

they are the same