sequencing + PCR

describe process of Sanger sequencing

run 4 sequencing reactions at the same time using forward primers, DNA template, DNA polymerase and normal dNTPs but also add ddNTPs to each reaction (1 reaction has ddATP, another has ddGTP, etc) and each is going to make strands of varying lengths and you can run a gel and see what each frag ends with and can do that to sequence

improvements made to Sanger

use fluorescently labelled ddNTPs so you can do a single reaction and separate fragments using gel electrophoresis

2 challenges to Sanger

need to know the starting sequence to design a primer, and the short genomic fragment has to be incorporated into plasmid for sequencing

describe pyrosequencing

DNA fragment is attached to solid surface microarray and flooded with a specific dNTP at a time, released pyrophosphate when it polymerizes which releases light, intensity of light is how many dNTPs were added, Apyrase then degrades all the unused nucleotides and ATP

describe the process of illumina sequencing

adaptors flank DNA fragments and bend like a rainbow on the chip using complimentary sequencing. Add fluorescent dNTPs and 3’ blockers so it only adds 1, computer analyzes which dNTP was added, then deblocking agents allow for another round of synthesis, do this until the whole thing is sequenced, then form contigs

list 3 reasons for sequencing

genome sequence of a single organism

metagenomic sequencing

sequencing RNA transcripts to measure expression

list the optimal temps in PCR

initial denaturation - 95ºC

annealing - 45-68 but 52-58 is ideal

elongation - 68

final extension - 68

hold - 4-10

describe the polymerase used in PCR

DNA pol 1 from thermus aquatics (Taq pol) is thermostable or else you would need a new one after each denaturation, low fidelity and no proofreading

what are considerations when thinking about the type of DNA pol. to use

depending on application, what the template is, pH, MgCl2 (affects annealing temp), primers

how to calculate Tm

(4 x [G+C]) + (2 x [A+T]) ºC

forward vs reverse primers

forward - same as coding, comp to non-coding

reverse - same as non-coding, comp to coding, found by reverse compliment coding strand

requirements for designing primers

no more than 3 of the same bp in a row, no self-complimentary sequences, 3’ end should end in a G or C and not bind to any site on the complimentary partner primer

what should RT-PCR mean

reverse transcriptase

why do we use cDNA in PCR

DNA is more stable and RNA pol is not thermostable

why is quantitative PCR aka real time PCR

bc it monitors fluorescent labeling during the process

3 functions of RT-PCR

RNA-dependent DNA pol makes a DNA-RNA hybrid

RNase H degrades the RNA part of that hybrid

DNA-dependent DNA pol

what is CT

cycle threshold, in qPCR its the number of cycles for the fluorescent signal to pass the threshold, the higher the number the less starting template you have

2 main ways for qPCR

dye based and probe based

describe dye-based qPCR

usually uses SYBR green, non-specific and binds to any dsDNA meaning you have to be careful with primers bc if they can complimentary bind it’ll bind to it and count it as a dsDNA

describe probe-based qPCR

when dye and quencher are together, no signal, when sequence specific part is bound to the DNA, quencher and dye are far and there is a signal

what does relative qPCR measure and why do we do this

fold differences between genes in different conditions

used to correct how much/quality of RNA used in reverse transcription

what does the reference gene have to be in relative qPCR

has to have nearly identical expression in the conditions your testing

how to calculate ∆CT and ∆∆CT and fold change in gene expression

CT (target) - CT (reference)

if replicates would calculate average

∆∆CT = ∆CT (T) - ∆CT (R)

2 ^ (-∆∆CT)