nucleic acid extraction

use detergent for cell lysis

add enzymes to destroy undsired rna and proteins-rnase and protease as these can degrade and contaminate dna

precipitate protein with alcohol

cell lysis with detergent

add chemicals to prevent denaturing of rna(rnase inhibitory factors) and degrade undesired macromolecules and separate them from rna

organic extraction method: use phenol and then centrifuge

rna is precipitated out using alcohol

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solid phase extraction of nucleic acids-using spin column and centrifugation

cell lysis

absorption: nucleic acid bind to solid support(silica beads etc.) in presence of chaotropic conditions, buffers with specific pH and salt concentrations

washing of beads with alcohol to remove contaminants

elution-beads washed again with a liquid that frees the nucleic acid into the solution

high throughput method:magnetic bead dna extraction

polymerase chain reaction process

components

dna polymerase

pcr buffer

primers

pcr tube

dna polymerase

thermocycler

dna template

one cycle of amplification

initial denaturation at 94 to 98c for 2 to 5 min to denature dsDNA

denaturation, same temp for 0.5 to 2 min

annealing to join ssDNA to primer at 50 to 65c for 0.5 to 2 min

Extension-dna polymerase extend the primers, adding nucleotides to the 3' ends of each primer, forming new strand of dna from template dna

70 to 75c, time varies based on length of target dna and synthesis rate of dna polymerase

final extension-72c for 5 to 15 min, help to fill in incomplete ends, duration depend on amplicon length and composition

amplification consists of denature, annealing and extension stage, done 20 to 40 times

take note of direction of extension, 5' to 3'

primers become part of dna molecule

18 to 30 nucleotides, has a GC content of 40 to 60%,

3' end should be exact match to template and ending in c or g-promote binding, last 5 bases should contain at least 2 G/C

avoid segments with >3 consecutive repeated nuceltodies-can cause mispriming

avoid complementary sequences within and between primers-prevent self annealing with each other

tm-melting temp-2c x (A+T)+4c x (G+C)

provides an estimate for which the primer anneals tp template

too high, primer cannot anneal, low pcr yield

to low-primer binds non-specifically to template, non-specific pcr product formed

ad and dis advantages

ad

dis

rapid amplification, highly specific, sensitive, versatile

dna sequence needs to be known to design primer, prone to non specific amplification if cycling parameters not optimised, presence contaminants, taq polymerase no proofreading ability

reverse transcription PCR

rna strand is template

rna is revered transcribed into complementary dna using reverse transcriptase

enzyme which has the rnase function degrades rna portion after dna/rna hybrid is formed

ssDNA formed, polymerase function of reverse trnascriptase synthesis complementary strand

dsDNA formed

start normal pcr test

rna is an unstable molecule, short half life after extraction due to presence of rnases in blood, tissue, bacteria

advantage is its low price and convenience

disadvantage is that it cannot handle large sample volumes, inconvenient for high throughput method

nucleic cell analysis

amplify dna using pcr

amplify rna using rt pcr

amplifies a small segment of the dna to produce a million copies

fast and inexpensive

involves repeated heating and cooling to allow polymerase to generate copies

initial denaturation, amplification(denaturation, annealing, extension), final extension

cycle repeated ~20 to 40 times

dna replication occuring in vitro, old dna is replicated, with one strand being the parental and the other strand being the new strand

thermocycler-temp for dna replication and synthesis depends on sequence of dna and pcr buffer conditions

number of cycles is based on initial amount of dna and final amount needed for downstream visualisation and analysis

you need dna template, dna polymerase, primers, bases(dATP, dTTp, dGTP, dCTP), pcr buffer, pcr tube and thermocycler

7 items

one cycle of amplification

annealing-primers align and attach to the ssDNA template strands, done from 50 to 65c for 0.5 to 2 min

extension-polymerase extend primers by adding nucleotides to the 3 ends of the primer, forming new strand of dna from template dna, done at 70 to 75c, time depends on length of target dna and synthesis rate of polymerase

denaturation-dsDNA is denatured at 94 to 98c for 0.5 to 2 min

initial denaturation done at 94 to 98c for 2 to 5 min

final extension-72c for 5 to 15 min-fill in incomplete ends, duration depends on amplicon length and composition