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nucleic acid extraction - Coggle Diagram
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
rna is an unstable molecule, short half life after extraction due to presence of rnases in blood, tissue, bacteria
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
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
rapid amplification, highly specific, sensitive, versatile
dis
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
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
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
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
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
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
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
amplify rna using rt pcr