unit 3 molecular

purpose of isolation and extraction

Release nucleic acid from cells to be amplified, remove inhibitors and contaminates, high concentrate nucleic acids, stabilize nucleic acids, increase quality (free from other nuc acid) and quantity of nucleic acids

isolation methods depend on

specimen type, cost and ease, type of nuc acid needed, purity, product yield

dna rna extraction step 1

lyse cells- release nucleic acids using detergents, enzymes, or mechanical disruption

dna rna extraction step 2

nucleic acid separation- separate dna or rna from proteins, lipids, debris, and other contaminants by centrifuge, chemicals, or binding to solid surface

dna rna extraction step 3

purification- remove contaminants and inhib improving nucleic acid quality for downstream applications

dna rna extraction step 4

recovery or elution- collects pure dna or rna in a buffer or solution to produce nucleic acid for molecular testing

factors determining selection method

specimen type, dna or rna, required purity level, expected yield, ease, time, cost, automation level, downstream application

we detect nucleic acids in

blood, tissue, micro

specific lysis

Blood and bone marrow- heat up to 90 C

Bacteria and fungus- add enzymes or detergents

Tissue- glass particles grind up tissue

Fixed(adhered, block of parafilm or formalin) specimen- must break apart or unfix

lysis buffer- breaks open the cell to release its contents

precipitate

final step to bring rna or dna out, cold alc (ethanol or isopropanol) and salt, alc disrupts H binds releasing rna or dna from solution, centrifuge, take off supernatant on top, re suspend pellet on bottom in a buffer, buffer is slightly basic- like tris edta

dna isolation and extraction ORGANIC

classic chem method, cell lysis, phenol and chloroform separated molecules by solubility, dna stays in aqueous (water) phase, proteins and lipids move to organic phase on bottom, produces high quality dna, most pure, labor intensive and toxic

organic liquid

cell lysis, mix phenol and chloroform, proteins/lipids settle to bottom, centrifuged, remove top aqueous layer- nuc acid layer, precipitate dna with alc, rehydrate in buffer, use or store product, high yield of nuc acids but not best quality

dna isolation and extraction INORGANIC

cell lysis with SDS, Sodium acetate salts out or precipitate rna and proteins out of solution, dna stays in liquid phase, centrifuge, dna is precip with alc and rehydrated in buffer, simpler safer faster, less pure, larger sample volumes, less complete separation, residual contamination

liquid extraction

large sample volumes, large nuc acid quantities, incomplete separation, residual contamination, more manual less standardized, org and inorg

dna isolation and extraction SOLID

cell lysis, protein digestion, dna precipitated by alc and binded to solid matrix column of silica or mag beads to be purified, lysate is added to column helps binding / absorbing dna to solid phase, wash to remove contaminated, dna eluded in nuclease free water by buffer, recover pure dna, clinic lab method, automatable, multiple samples at once, consistent and reproducible, safer, best method, expensive

silica vs mag

silica is slower than mag faster than organic, less automated than mag but more than organic, more common but lower throughput than mag

rna isolation and extraction

has RNase control (protecting rna) but equitment PPE etc must be RNase free, separate nucleated cells from rbcs before lysis, denature proteins, separate and bind to column, ass DNase to remove dna, precipitate rna with alc, resuspend in buffer, must be quicker and minimal freeze thaw cycles

rna inhibitor

can add throughout whole process to inactivate RNase (guanidine phenol)

A student is performing an RNA isolation and skips the additional RNase control steps

The RNA will be degraded and the yield will be poor

What is the specific effect of a lysis buffer when added to a cell suspension?

break open cell releasing its content

in inorganic liquid phase extraction, what reagent is added specifically for protein precipitation

sodium acetate

spectrophotometry basics

most common, absorbance to determine purity, quality and quantity, precise and accurate, easy, cheap, fast

spectrophotometry absorbance of UV- PURITY

dna/rna 260 nm

protein 280 nm

purity = abs at 260/ abs at 280, 1.8 for dna, 2 for rna, if low (230) contamination by salt carbs phenol or guanidine

spectrophotometry absorbance relationship to amount of acid and quality

proportional, but high abs lower quality

phenol

abs at 270 which is similar to dna and mistaken by spectrophotometry

spectrophotometry dna quality/purity

1.8, 1.6-2, low = protein or phenol contamination, high = rna contamination or measure error

spectrophotometry rna quality/purity

2, 1.8-2.3, low = protein or phenol contamination, high= degradation

1 mL of blood =

30-60 ug of nuc acid

spectrophotometry quality concentration beer-lambert law

abs at 260 × 50 for dna or 40 for rna x dilution factor x volume prepared

yield

concentration x volume

electrophoresis

separates by molecular weight (size) and charge, confirming amplification, analyze fragment size with dna ladder to assess nuc acid integrity

rules of electrophoresis

nuc acid neg charge by backbone migrates toward anode+ (smaller faster towards the bottom), stain, detects cancers by testing for Ig in sample, intensity and brightness = quantity/quality (light= degradation)

high quality genomic dna on electrophoresis

single high molecular weight band, minimal smearing

degraded dna on electrophoresis

smearing, few intact large fragments, due to poor pcr amplification or reduced sequencing quality

degraded rna on electrophoresis

smearing, weak or missing rrna bands, produces unreliable downstream results

high quality rna bands electrophoresis

28S and 18S,= 2:1 intensity ration

electrophoresis applications

confirm successful restriction enzyme digestion, detect genomic dna contamination, verity pcr products

electrophoresis limits and pro

semi quantitative and labor intensive, but goood for dna/rna quality

fluorometry

dyes bind specifically to dna/rna, accurate, emits fluorescence when excited by ligh wl, intensity proportional to nuc acid concentration, selective and sensitive (quantification not quality or purity)

fluorometry application

low concentration samples, prepping samples for sequencing, prc, or qpcr

fluorometry limits and pro

requires standards and specialized reagents, no purity, BUT accurate quantification before downstream applications

troubleshooting

poor quality= repeat isolation of sample, concentrate nuc acids with alc precipitation

low yeild= repreat isolation of sample, clean up by re- isolation