3 - Nucleic Acid Extraction and Quantification

Typical workflow

• Request for genetic testing

• Sent to lab (blood, buccal, amniotic, fetus)

• DNA extraction for microassays, PCR, sequencing

• Culture for cytogenetic testing - karyotyping, FISH

Sample and testing types

• Molecular oncology - biopsies, surgical specimens, circulating cell free tumour DNA (ctDNA)

• Non invasive prenatal testing (NIPT) - circulating cell free fetal DNA (cffDNA) from maternal plasma

• Infectious disease testing - urine, stoop, sputum

• Germline testing - whole blood, saliva

Whole blood, serum, plasma and bone marrow aspirates

• Collected in anticoagulant / additive tube - depends on test performed and sample collected

• Heme and heparin are PCR inhibitors

• If RNA is tested, an RNA stabilising additive must be used, degradation ex vivo

Prenatal specimens

• Can identify genomic alterations associated with genetic disorders, NIPT

• Chorionic villus sampling or amniotic fluid

• cffDNA isolated, 3-10% of circulating DNA, collected at 10-22 weeks

Tissue specimens

• Can be fresh or stabilised (frozen, fixed)

• Factors that affect results - cold ischaemia time (<1h), type of fixative, timein formalin (>24h), specimen thickness

• May need to be dissected, specimens containing inadequate neoplastic cells should be rejected

Cervical, endocervical, vaginal and urethral specimens

• Male urethral collected with swabs

• Female collected with swabs and placed in appropriate medium

Urine and stool specimens

• Collected in clean, sterile container, stored at 4c

• Some STD assays need specific collection devices

• Stools should ideally be fresh or transferred to container with fixative compatible with molecular testing

Sterile body fluids

• CSF, pleural fluid collected using aseptic technique

• No cathaters or shunts - commensals

• Respiratory specimens - nasopharyngeal swabs, bronchial lavages, follow assay directions as type affects results

Isolating and purifying nucleic acids

• Performed with nuclease free water, molecular grade reagents

• Manual - flow through columns, automated column or magnetic

• Complete specimen distruption and cell lysis essential, lysis buffer with detergent and protease

• Low cellularity specimens centrifuged (CSF)

• Heating stool samples may increase yield from hard to lyse microbials (gram positive bacteria)

• Mechanical force (sonication) used in respiratory samples

Nucleic acid isolation methods

• Chemical - phenol-chloroform

• Extraction kits - silica based column or magnetic bead purification

Silica membrane method

• DNA and RNA isolation, high throughput, can be manual (microcentrifuge) or fully automated

• Positively charged silica membrane in presence of a high salt concentration absorbs negatively charged nucleic acids

• Chaotropic salts lyse cells and deactivate RNAses

• Ionic strength of lysis and binding solutions can be adjusted for selective binding (DNA or RNA)

• Debris washed away, nucleic acids eluted using low salt buffer

Magnetic particle method

• Manual and automated (MagNA pure 96)

• Magnetic beads coated with silica particles without centrifugation

• Coated with specific biopolymers with affinity for DNA or RNA

• Once nucleic acids bond a magnet is applied to pull the particles out fo solution

• Impurities washed away, nucleic acids eluted, magnetic particles removed using magnet

Nucleic acid elution

• Eluted in nuclease free water, TE buffer or commercial buffer

• RNA may be co-purified with DNA, treated with RNAse A (heat treated first to destroy DNAse)

• DNA removal may be important fot some assays (BCR/ABL, RT-PCR), gDNA digested with DNAse 1

Nucleic acid storage

• Hydrophobic plastic tubes with rubber gaskets to prevent evapouration

• Stored in solution as above

• RT for 6 months, 2-8c for 1 year if DNAse free, -20 to -70 for years

• RNA stored at -70, aliquot to avoid freeze thaw cycles (degredation, fragmentation)

• No frost free freezers (cycles)

Evaluating nucleic acid purity using spectrophotometry

• NanoDrop - microvolume (0.5-2ul), sensitive

• Nucleic acids absorb light at 260 through adenine residues, directly peoportional to concentration of sample (1 = 50ug/ml dsDNA)

• 260/280 ratio, pure DNA is 1.8, pure RNA is 2, protein will reduce ratio

• 260/230 ratio indicated contaminants that absorb at that wavelength e.g. chaotropic salts, EDTA, phenol

• Pure DNA is 2.3-2.4, pure RNA is 2.1-2.3

Fluorometric methods

• Qubit - measured flourescence related to nucleic acid concentration

• Optimised dyes bind to target on DNA or RNA and only emit signal then

• Concentration obtained by comparing relative flourescence units of the sample to those of standards

Electrophoresis methods

• Visualise, quantify and qualify nucleic acids, and provide size of product

• Flourescent dyes e.g. SYBR safe or gel red, intensity related to quantity

• RNA - two bands will appear, if bands are degraded or absent the quality is deemed unsuitable for molecular assays

Fragment analyser (automated)

• Parallel capillary electrophoresis e.g. agilent 5200

• Performs QC for large range of sampels - gDNA, cfDNA, RNA, mRNA

• Quality metrics - genomic quality number, DNA quality number (1-10), RNA quality number (1-10), degraded vs intact

Tapestation systems

• Detect flourescently stained dsDNA and total RNA

• ScreenTape device detects DNA fragments, genomic DNA, cell free DNA, total RNA

• Has 16 lanes, eliminates contamination

• QC metrics - DNA and RNA integrity numbers