NAME: ____________________________________________________ CODE/SCHED: ___________

MLS325L: MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY

ACTIVITY 02: NUCLEIC ACID EXTRACTION

OBJECTIVES:
1. Identity the preanalytical variables that must be considered in the performance of nucleic acid
extraction
2. Provide the rationale for the conduct of crucial steps in nucleic acid extraction
3. Perform a specific nucleic acid extraction protocol
4. Assess the quantity and quality of extracted nucleic acid sample

INTRODUCTION:

TYPICAL GENOMICS DIAGNOSTICS WORKFLOW

TARGET GENOME
CHARACTERIZATION
NUCLEIC ACID REGION
OF AMPLICON/
EXTRACTION AMPLIFICATION/
SEQUENCE ANALYSIS
HYBRIDIZATION

SPECIMEN TYPE
 Depending on the purpose of the molecular test for which the nucleic acid sample is to be
extracted, some specimen types are preferred over others.

Table 1. Examples of preferred specimen for PCR diagnosis per pathogen

Pathogen Preferred Specimen for Testing
MERS- Coronavirus  nasopharyngeal swab and oropharyngeal swab in virus transport medium or
universal transport medium
 nasopharyngeal aspirate
 sputum
 endotracheal aspirate
 bronchoalveolar lavage
 post-mortem lung tissue (at least 3cm3)
Influenza virus  nasopharyngeal swab and oropharyngeal swab in virus transport medium or
universal transport medium
 nasopharyngeal aspirate
 sputum
 endotracheal aspirate
 bronchoalveolar lavage
 post-mortem lung tissue
Ebola virus  serum (yellow-top)
Zika virus  serum (yellow-top)
 CSF
 urine
 amniotic fluid
 saliva

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Malaria  whole blood in EDTA tube

 dried blood spot
Varicella Zoster Virus  tissues
 vesicular fluid
 crusts from lesions
 cerebral spinal fluid (CSF)
 blood
Cytomegalovirus  blood
 congenital CMV infection: saliva, with urine usually collected and tested for
confirmation
Epstein Barr Virus  blood and CSF
HIV Viral Load,  whole blood in EDTA tube
Hepatitis C Virus
RNA and Hepatitis B
Virus DNA
Neisseria  urine
gonorrhoeae &  endocervical swab
Chlamydia  urethral swab
trachomatis  oral or pharyngeal swab
 rectal swab

SPECIMEN COLLECTION AND HANDLING

 The accuracy of molecular test results is highly dependent on the quality of the specimen.
 Hence, molecular tests require optimal specimen handling and processing for accurate and
consistent test results.
 The main consideration in handling and processing specimens for molecular biology is to
prevent contamination as: (i) the quality/quantity of nucleic acid extracted is greatly impacted
by contaminants that may be present before and during extraction; and, (ii) PCR and other
amplification techniques are extremely susceptible to contamination problems.

1. Specimen collection
 Timeliness must be a priority in doing specimen collection.
 For molecular testing of infectious agents:
o The time and site of collection must be optimal – relate to microorganism’s
pathogenesis or disease progression
o The type of specimen and collection time must be appropriate for the target
pathogen to be detected.
o Respiratory tract specimens should be collected as soon as possible in the course of
the illness and before antibiotic therapy begin, if possible.
o The likelihood of recovering most viruses and many bacteria diminishes markedly
>72 hours after symptom onset and after the initiation of appropriate antimicrobial
therapy.
o If possible, respiratory specimens should be collected within 72 hours of symptom
onset and no later than 7 days after onset.
 The quantity per specimen type must also be considered.

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Table 2. Required quantity of specimen for molecular testing

Specimen Quantity
Whole Blood 3ml – 5ml
Serum 1ml – 2ml
Urine (Leptospirosis) 15ml
CSF 0.5ml – 2ml
Sputum >1ml
Oropharyngeal lesion 2 Dacron swabs
Nasopharyngeal swab 2 Dacron swabs (left and right nostrils)
Stool 5g -10grams Formed
5ml – 10ml Watery
Tissue >1cm
ITCF >200ul

2. Specimen handling
 A requisition or electronic test order should accompany the specimen.
 If a specimen shows evidence of tampering or is hemolyzed, degraded, clotted, or
otherwise compromised, the technologist must notify the supervisor.
 No specimen is accepted without proper labeling and identification on the specimen tube
or container (placed by the person who collected the specimen), nor is a specimen
accepted if the labeling on the specimen does not match that on the accompanying
requisition.
 The following are some additional criteria for specimen rejection:
o Specimen quality (Consequence: Request for a new sample from the
patient/requisitioner/referring institution.)
 Inappropriate specimen type
 Leaking container
 Insufficient quantity
 Suspicion of contamination
 Inappropriate transport or storage
 Unknown time delay
o Specimen information (Consequence: Specimens may be processed but results
will not be released until the missing information is supplied by the
requisitioner.)
 Missing or incomplete laboratory request form
 Minimum information required: (full name, age, requisitioner’s name
and contact number)
 Unlabeled specimen
o Referral coordination (Consequence: Possible delay of result or complete
rejection of specimen)
 Testing laboratory is not notified of the parcel shipment.
 No transmittal letter prior to receipt of specimens.
 Test requisition should include the following information:
o Relevant patient identification
o Type of specimen material (e.g., blood or bone marrow)
o Ordered test

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o Date and time of collection

o Reason for testing
o Contact information of the ordering physician
o For molecular genetics, forensics, or parentage testing, the supplemental
information in the request form include:
 Patient consent forms
 Ethnicity
 Photo identification of the individuals tested
 Patient label verification
 Transfusion history or a pedigree
 Forensic specimens may require a documented chain of custody.
 Hemoglobin inhibits enzyme activity. Specimens received in the laboratory should
therefore be inspected for visual signs of hemolysis.
o In routine DNA and RNA isolation procedures, red blood cells are lysed followed by
removal of hemoglobin.
o However, if white blood cell lysis has also occurred, DNA or RNA yield will be
reduced.
o Buffers and resins have been designed to sequester anticoagulants or hemoglobin
for more rapid nucleic acid isolation without the inhibitory effects of these
substances.
 Solid tissues are best analyzed from fresh or frozen samples, especially for Southern blot
or long-range polymerase chain reaction (PCR) methods that require relatively high-quality
(long, intact) DNA.
 Surgical specimens designated for molecular studies, if not processed immediately, should
be snap-frozen in liquid nitrogen.
o Snap freezing is routinely performed in the surgical pathology laboratory because
it is a common process for preserving tissue morphology for microscopic
examination.
 Fixed, paraffin-embedded tissues generally yield lower-quality DNA and RNA
o PCR and reverse transcription PCR (RT-PCR) amplification are routinely performed
on paraffin-embedded tissue samples.
o Methods such as Southern or northern blot, requiring large fragments of DNA, are
less likely to work consistently with fixed tissues.

3. Collection tubes for molecular testing

 For molecular analysis, blood or bone marrow specimens collected in EDTA (lavender-cap)
or ACD (yellow cap) tubes are preferred.
 Heparin (green cap) is used for cytogenetic tests.
o Heparin has been shown to inhibit enzymes used in molecular analysis, such as
reverse transcriptases and DNA polymerases in vitro.
o The influence of heparin inhibition on molecular analysis is commonly accepted
though but should be noted in interpreting the results.
o Interference by heparin is also affected by assay design. It should be noted that
heparinized samples have been processed successfully in many laboratories.

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Table 3. Evacuated tubes for molecular testing

Additive Color Nucleic Acid Testing
Sodium citrate Light Blue/ Black Top Molecular diagnostic testing
(Tube also with polyester gel (Cell Preparation Tube) **Mononuclear cells and platelets are
and density gradient liquid) separated from the granulocytes by the
polyester gel and density gradient liquid
Sodium heparin Red/Green Top Molecular diagnostic testing
(Tube also with polyester gel (Cell Preparation Tube)
and density gradient liquid)
None Red Chemistry, serum, viral antibody studies
Sodium heparin Green Immunology, Virology studies
(freeze-dried)
Sodium heparin Brown Cytogenetic studies, molecular studies
Tripotassium EDTA Lavender Virology, molecular biology studies
(7.5%-15% solution)
Acid citrate dextrose (ACD) Yellow Molecular biology studies
solution Cellular studies, Human leukocyte antigen
(HLA) phenotyping, Paternity testing

4. Specimen storage

Table 4. Storage requirements for specimens to be subjected for nucleic acid extraction
Nucleic Acid Sample Temperature Time
22-250C 24 hours
Whole blood, buffy coat, bone 2-80C 72 hours
marrow, fluids -200C At least 1 year
-700C More than 1 year
22-250C Not recommended
2-80C Up to 24 hours
Tissue
-200C At least 2 weeks
DNA
-700C At least 2 years
22-250C 24 hours
2-80C 72 hours
Microorganisms in culture
-200C 2-4 weeks
-700C More than 1 year
Cell lysates in guanidine
22-250C 1-2 weeks
isothiocyanate (GITC)
22-250C Not recommended
Whole blood, buffy coat, bone 2-80C 2-4 hours
marrow, fluids -200C 2-4 weeks
-700C More than 1 year
Fluids collected in specialty 22-250C 5 days
2-80C 7 days
RNA
RNA protection tubes -200C 2-4 weeks
-700C At least 7 months
22-250C Not recommended
2-80C Not recommended
Tissue
-200C Not recommended
-700C At least 2 years

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-1400C (nitrogen vapor) At least 2 years

Cell lysates in guanidine
22-250C 1-2 weeks
isothiocyanate (GITC)
22-250C 1 week
2-80C 1 month
Cell lysates in RNA storage -200C More than 1 year
solution (Ambion) 2-80C 72 hours
-200C 2-4 weeks
-700C More than 1 year

 After collection, extracted nucleic acids should be properly stored if they are not to be
processed immediately.

Table 5. Nucleic acid storage requirements

Nucleic Acid Matrix Temperature Time
0
TE buffer or DNAse-free water 22-25 C Up to 4 months
Freeze-dried or dried on collection paper 22-25 0C More than 15 years
DNA TE buffer or DNAse-free water 2-8 0C 1-3 years
TE buffer or DNAse-free water -20 0C At least 7 years
TE buffer or DNAse-free water -70 0C More than 7 years
TE buffer or RNAse-free (DEPC-treated)
22-25 0C Not recommended
water
TE buffer or RNAse-free (DEPC-treated)
2-8 0C Not recommended
water
TE buffer or RNAse-free (DEPC-treated)
-20 0C Up to 1 month
water
RNA
RNA storage solution (Ambion) -20 0C More than 1 month
0
Ethanol -20 C More than 6 months
TE buffer or RNAse-free (DEPC-treated)
-70 0C Up to 30 days
water
Ethanol -70 0C More than 6 months

PRECAUTIONS AND DECONTAMINATION

 Generally, RNA is less stable than DNA primarily because the 2’-OH on RNA makes it more
prone to hydrolysis in basic conditions, making it more susceptible to degradation when
compared to DNA.
 Nucleases – very stable enzymes that catalyze the digestion of nucleic acids by cleaving
the phosphodiester bonds between nucleotides
o DNases – specifically digests DNA
o RNases – specifically digests RNA
 These enzymes are nearly ubiquitous, commonly found on human hands, bodily fluids,
contaminated water & reagents, laboratory instruments and surfaces, and in the sample
itself.
 With these in mind, several precautions should be undertaken when extracting nucleic
acids:

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1. Wear proper PPE, not only to protect yourself from the sample but also to prevent
contaminating the sample.
2. Designate work areas with controlled air flow solely for RNA and DNA extraction to reduce
nuclease contamination.
3. Decontaminate laboratory surfaces, equipment and instruments with nuclease inhibitors,
10 % bleach, and 70 % ethanol.
4. Use nuclease-free water and reagents, and treat polypropylene tubes and tips with
dimethyl pyrocarbonate (DMPC)/ diethyl pyrocarbonate (DEPC) to inactivate RNases.
5. Minimize the number of freeze-thaw cycles, since freezing and thawing changes the pH of
the sample, leading to nucleic acid degradation.

DIFFERENT EXTRACTION METHODS

1. Crude Lysis – involves breaking open cells by different means, but without any means to
isolate nucleic acids from the crude lysate
2. Liquid-Phase Organic Extraction – uses phenol and chloroform to separate hydrophilic
nucleic acids from hydrophobic lipids and proteins before precipitating nucleic acids out
of the solution with alcohol
3. Liquid-Phase Inorganic Extraction – uses salts to precipitate out proteins prior to
precipitating nucleic acids out of the solution with alcohol
4. Solid-Phase Extraction
 Uses a positively-charged silica membrane to filter out unwanted molecules and
bind negatively-charged nucleic acids under high salt conditions that can be filtered
out separately to yield pure nucleic acid
 Most commonly used for nucleic acid extraction kits
Involves 4 basic steps:
A. Lysis – cells are lysed using a chaotropic agent or detergent to release nucleic
acids
B. Binding – nucleic acids are bound to the silica membrane
C. Washing – unwanted molecules are filtered out while nucleic acids remain bound
to the silica membrane
D. Elution – nucleic acids are released from the silica membrane and filtered out
 Can also be automated in order to reduce the need for manual work
5. Magnetic Beads
 Uses magnetic beads that can bind nucleic acids, which can then be separated from
the mixture and treated to release the bound nucleic acids
 Can also be automated

DNA VS. RNA EXTRACTION

 RNA is less stable than DNA due to differences in their structure
 As such, stricter observance of the precautions mentioned above is required to prevent RNA
sample degradation

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PROCEDURES:

Bacterial DNA Extraction Protocol (tick off each step as you finish it)

 1. Pipet 1mL bacterial cell suspension into a 1.5mL microcentrifuge tube. Centrifuge for 1
min at 13,000g. Discard supernatant.

 2. Add 100µL EL buffer. Mix for 10 seconds. Incubate at 37oC for 20 minutes.
 3. Add 100µL RS buffer and 10µL PK solution. Mix for 10 seconds. Incubate at 56oC for 20
minutes.

 4. Add 200µL GA buffer. Mix for 10 seconds. Centrifuge for 1 min at 13,000g.
 5. Transfer supernatant to a new 1.5mL microcentrifuge tube.
 6. Add 400µL BA buffer. Mix for 10 seconds.
 7. Transfer mixture to spin column. Centrifuge for 1 min at 13,000g. Discard flow-through.
 8. Add 500µL GBinding buffer. Centrifuge for 1 min at 10,000g. Discard flow-through.
 9. Add 500µL Washing buffer. Centrifuge for 1 min at 10,000g. Discard flow-through.
 10. Repeat step 9.
 11. Spin dry for 1 minute at 10,000g.
 12. Transfer spin column to a new 1.5mL microcentrifuge tube.
 13. Add 100µL Elution buffer. Incubate at room temperature for 1 minute. Centrifuge for 1
min at 10,000g. Store eluted DNA properly.

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Extraction Assessment
 A good nucleic acid extraction protocol should meet the following criteria:
o Efficient extraction
o Sufficient amount of extracted nucleic acid for downstream applications
o Able to remove contaminants
o Yields highly pure nucleic acid
 One useful technique that can be used to assess these criteria is spectrophotometry
o A260 – quantitates RNA, ssDNA, and dsDNA
o A260/A280 – measures purity of nucleic acids
 Pure DNA = 1.8
 Pure RNA = 2.0
 Lower ratio = presence of contaminants
o A260/A230 – another measure of purity, with pure nucleic acids = 2.0-2.2

***For submission – group report containing the following:

A. Table containing the steps in the protocol in the first column and the rationale for performing
them in the second column
B. Spectrophotometric assessment of extraction protocol with discussion (i.e. A260/A280 reading
and reason/s why it is so with recommendations for improvement if needed)
C. Photodocumentation by step with name of performer included in the caption

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