Lab - Modx (Prelim)
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erikaadriennedavid04Lab - Modx (Prelim)
Uploaded by
erikaadriennedavid04Molecular Biology & Diagnostics
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science
PRELIM
Autoclave:
MOLECULAR LABORATORY SAFETY GUIDELINES ● used for sterilizing equipment used in the laboratory
https://www.youtube.com/watch?v=srURYFGb10k
MOLECULAR BIOLOGY Micropipette:
● used tom easureoutandtransfers mallquantitiesofliquid
GENERAL CHARACTERISTICS in m icroliters or m illiliters accurately; s hould be used in
● ranchofbiologythatdealswiththes tructureandfunctionof
B aseptic conditions
the macromolecules Micropipette tips:
● Responsible for the development and performance of ● disposable, autoclavable tips used to protect the
m olecular diagnostic test for nucleic acid m icropipette from contamination but it m ust be disposed
● target found in a variant of settings each use
EQUIPMENT P10 dispenses 0.5-10ul(white)
Thermocycler: P20 dispenses 2-20ul (yellow)
● apparatusthatisusedtoamplifys egmentsofnucleicacids P200 dispenses 20-200ul (yellow)
and has the capacity to change temperature based on its P1000 dispenses 200-1000ul (blue)
pre-programmed steps P5000 dispenses 1000-5000ul (white)
UV Transilluminator: Other materials
● allows the visualization of target DNA and proteins by ● Spatula,
emitting high levels of UV radiation through the viewing ● Baker’s cup,
s urface. ● Graduated conical tubes,
Gel electrophoresis chamber: ● Freezer boxes,
● used to isolated DNA or protein fragmentsbys izeinagel ● Ice buckets
m adeofagaroseorpolyacrylamidebythehelpofanelectric
charge REAGENTS
Spectrophotometer: LIST OF REAGENTS USED FOR MOLECULAR LABORATORY :
● m easures light intensity as a function of a wavelength to Tris-base Glacial acetic acid
m easure the concentration of a compound in aqueous NaCl (salt) Proteinase K
s olution MgCl2 Restriction enzymes
Microcentrifuge: EDTA powder HCl NaOH
● used for s pinning s amples at high-speed that allows Gel loading dye Boric acid
s eparation ofs olutionsbasedondensitys uchasenabling Molecular weight markers Sodium acetate
the pelleting of nucleic acids or proteins from solutions Absolute ethanol PCR kit
Microcentrifuge tubes: Nuclease free water ELSIA kit
● s pecially engineered test tubes that can resist high Distilled water DNA isolation kit
gravitational force
Refrigerated centrifuge:
STORAGE OF BUFFER AND SOLUTION
● used for s amples that need a consistent range of ROOM TEMPERATURE 4C
temperature
● Detergent ● Bacterial culture
Vortex mixer:
● Ethanol ● PCR products
● used for m ixing laboratory s amples, for agitation and ● Buffers ● DNA samples
encouragereactionsorhomogenizationwithhighdegreeof ● Concentrated solution ● Media
precision ● Acid and bases ● Serum
Shaker: ● Buffer
● used tom ixoragitates olutions,oftenpriortoplacementin -20C -70C
the centrifuge ● Enzymes ● Bacterial culture
Analytical Balance: ● RNA samples ● Lipids
● designed for accurate measurement of mass ● Always refer to the MSDS of each reagent to know its
Dry bath or water bath: composition and properties on how to proper handle
● used to heat sample appropriate for their incubation period especially when spilled and if for disposal.
pH meter:
● anelectricdeviseusedtom easurehydrogenionactivityofa
s olution
Biosafety cabinet:
● primary device used for containing infectious microorganism
● utomated m ethod usually uses m agnetic bead extraction
A
m ethods
LABORATORY PROTOCOL Performance of Molecular Tests
● Check all the parameters that s hould be within the
PREANALYTICAL STANDARDS acceptable limits
Reduction of Identification errors ● Confidence intervals(CIs)isusedtodeterminetheassays
● Patient and specimen compatibility performance
● pecimen identification: checking of label, fixative used,
S OSTANALYTICAL PHASE
P
transport date and time Molecular Diagnostic Report
Sample Receipt and Handling ● Should convey accurate information
● Accessionnumbers:thisistoensuretheproperhandling ○ Patient identification
ofeverysampleinaccordancew iththeISO15189andISO ○ Reporting style and content
9001 guide ■ One page of single screen reports is preferred
■ Name ofindividualtakingresponsibilityofthetesT
● pecimen should be handled properly based on their
S and contact details is important
required standard operating procedure ■ There s hould be s pell checkers to prevent
○ Tissuebiopsys ample:canbereceivedineitherfixedor typographical errors
fresh sample ○ Interpretation
○ Tissue blocks and s lides: s tored in ■ It m ust be reported correctly and interpretation
temperature-controlled environment s hould be provided
○ Blood s ample: received in the required anticoagulant ■ Genotyping results s hould be given according to
and discarded within few days Human GenomeVariationSocietynomenclatureat
● Minimization of cross-contamination the DNA level
○ Replace the knife blades regularly especiallyforFFPE
block
REPORT INFORMATION
○ The use of water baths to s tretch s ection can be
avoided by using a droplet of PCR- quality water on egistration
R number, age,
Patient Identification
glass slides gender, ordering physician
○ Collection time and date s hould be indicated in the Formalin-fixed paraffin
Specimen Type
s pecimen embedded, fresh, frozen
○ Specimen processing s hould be done within 30 Pathological Diagnosis Name of disease
m inutes Specimen number, block
Tissue sample Identification
● Specimen Requirements number
○ Criteria s hould be m ade for FFPE, fresh tissue, Important dates Date on reception or report
blood/bone m arrow, cell-free DNA for s pecimen NGS Method Molecular method used
acceptance that includes both quality and quantity of Interpretation
s pecimen
○ Storage of DNA and RNA
Sample results form:
■ Extracted DNA and RNA sample: -20C or lower
■ PCR products: -20C or lower
■ Sequencing libraries sample: -20C or lower
● Choice of analytical methods
○ Considersthevolume,diagnosticyieldandfeasibilityof
performingthetestinthelaboratorys ettingsthatiscost
effective to consumers
○ Factors determining thefeasibilityincludesthetraining
required,turnaroundtimeofthetesttoperformandthe
purpose of the test
ANALYTICAL PHASE
DNA and RNA Extraction
● Majority of incubation s teps m ust be kept at lower
temperature to inhibit possible nuclease activity while
s toring DNA or RNA samples on ice
● It is recommended to do initial DNA damage repair for
genomic DNA sequencing applications
● Test kits s hould be validated and m ethods s hould be
verified in performing the procedure
● Manualm ethods houlduseaprecipitationwashs tepswith
centrifugation of spin columns
M olecular Biology & Diagnostics M AKATAK 2
MATERIALS AND EQUIPMENT IN MOLECULAR DIAGNOSTICS MICROCENTRIFUGE TUBES
● Aka Eppendorf tubes
PERSONAL PROTECTIVE EQUIPMENT (PPE) ● Tapered,s peciallyengineeredtesttubesforcentrifugework,
● SAFETY GLASSES/GOGGLES designed to resist high G-forces induced by centrifugal
● GLOVES forces
● LABORATORY GOWN/COAT MIXERS
● LABORATORY FOOTWEAR VORTEX
SEROLOGICAL PIPETTES ● Stirs at controlled speeds to mix liquids
● Toaccuratelypipetterelatives mallvolumes,typically1-25m l SONICATOR
during experimentation ● Applies sound energy, used to fragment molecules of DNA
● Individuallywrapped,s terilepipettesareusedwhenworking ● Protein expression and purification
with cell cultures under aseptic conditions ● Uses:
MICROPIPETTES ○ Mix cells in suspensions
● Instrumentusedtom easureandextractverys mallamounts ○ Mix reagents of an assay to create homogenous
of liquids from a solution s olutions
● Differenttypeshavedifferentlevelsofaccuracy,butusuallyto VORTEX MIXER SONICATOR
the nearest microliter (uL) WATER BATH
● Often used to handle s mall amounts of s olutions in ● Container that uses water to heat or m aintain a constant
m olecular biology temperature in a
PLASTIC MICROPIPETTE TIPS ● highly controlled manner
● Disposable, autoclavable tips used to protect the ● Uses:
m icropipetter from contamination ○ To heat or thaw fragile biological s ubstances like
● Used and disposed after each use in order to prevent m ammalian cells
contamination and reduce sterile clean up and sterilization ○ To m aintain a m ore homogenous temperature control
AUTOCLAVE when working with nucleic acids and proteins
● Removes m icroorganisms (virus, bacteria, fungi, etc) and WATER BATH DRI BATH
s pores using high pressure and high temperature s team SHAKER
s terilization ● Usedtom ixoragitates olutions,oftenpriortoplacementin
● Sterile contaminated materials like biohazardous wastes the centrifuge or spectrophotometer
● Sterile glassware, plastics and pipettes for use in aseptic ● Uses:
techniques ○ To incubate antibodies, chemical reactions during
● Sterile liquids for use in experimentation experimentations for mixing and reproducible results
CENTRIFUGE ○ To aerate m edia while growing bacterial or yeast
● Is an apparatus that rotates at high s peed and s eparates cultures
s ubstances of different densities CO2 INCUBATOR
○ Microcentrifuge ● Sealed chamber with controls tom anagetemperatureand
■ Lightweight, s mall volume (0.2-2.0 m L), fast s ometime humidity and CO2 levels
acceleration for s hort intervals, uses Eppendorf ● Used to culture adherent cells under s pecific conditions
tubes m imicking normal or diseased in vivo environment
○ Ultracentrifuge PETRI DISHES/FLASK
■ Isolates mallerparticles,plasmids,DNA,RNAand ● Typeofglassorplastics hallowrounddisk/flaskcoatedwith
proteins a extracellular matrix (ECM) like material
● Uses: ● Uses:
○ Remove cell elements from blood to provide cell free ○ To culture mammalian cells in vitro
s erum or plasma ○ Can also hold culture m edium/agar to grow bacteria
○ Separate s ubcellular components (mitochondria, and viruses
nuclei, etc) MICROPLATE READER
○ Remove insoluble matter from extracted proteins ● Aka plate readers or microplate photometers
○ Pellet, DNA, RNA, proteins and lipids in isolation of ● Allows a variety of tests to be performed and measured
m acromolecules ● Multiwell plates (microplates)
MICROCENTRIFUGE ULTRACENTRIFUGE ● Uses:
MICROSCOPES ○ Quantify protein, gene expression and various
● Used to visualize objects and areas of objectsthatcannot m etabolic processes
be seen by the naked eye ○ Samples are measured by:
○ Fluorescence microscope ■ Absorbance – ELISA, protein and nucleic acid
■ Target proteins, cells or cellular components quantifications, enzyme activity assays
■ Uses fluorescent dyes/fluorochromes ■ Fluorescence–more expensive, more sensitive
○ Electron microscope ■ Luminescence–Optically simpler the fluorescence
■ Higher resolution (up to 100,000 magnification) pH METER
■ Cannot be used on live samples ● Indicates the acidity or alkalinity of as olutionexpressedin
M olecular Biology & Diagnostics M AKATAK 3
pH ● igh pressure liquid chromatography(HPLC)–purification
H
Low pH –acid solution (pH <7)
● of m any m olecules within a s hort time (carbohydrates,
● High pH –basic solution (pH >7) lipids, proteins, nucleic acids)
BIOLOGICAL SAFETY CABINET
● Primary containment of microorganisms
● ATP, dCTP, dGTP, and dTTP
d
● Acts as barriers ● RUTH ELLEN ALONZO10:47 AM
● Minimize the risk of airborne infections ● deoxyadenosine triphosphate (dATP), deoxythymidine
● Primary purpose protect the laboratory personnel and the triphosphate (dTTP), deoxycytidine triphosphate (dCTP),
environment from pathogenic microogranisms deoxyguanosine triphosphate (dGTP)
● With HEPA and ULPA filters
● Have different classes: Class I, II, and III BSC’s
FUME HOODS
● Usedtocontainhazardousgases,vapors,m ists,aerosols,
and paraticulates
SPECTROPHOTOMETER/SPECTROMETER
● Produceslightofdesiredwavelengthanditpassesthrough
yours ampleandreachesthephotometerthatm easuresits
intensity
● Reports the value as amount of light absorbed (ABS)
● Uses:
○ Measure growth of m icroorganisms like bacteria and
yeast or other cells grown in suspension
○ Find concentration of proteins or nucleic acids
○ Detect contaminants
ELECTROPHORESIS APPARATUS
● Separate DNA, RNA,andproteinm oleculesbasedontheir
s ize and electrical charge
● Gel matrix to sieve into pieces according to sizes
● Components of the apparatus:
○ Gel box, wells, comb
○ Electrodes – provide current, positive and negative
electrodes
○ DNA binding dye – to visualize DNA/RNA or protein
bands separated by gel electrophoresis
GEL ELECTROPHORESIS CHAMBER
● Used to isolate DNA or Protein fragments by s ize in agel
m ade of agarose (DNA/RNA) or Polyacrylamide (proteins)
● The electrophoresis chamber has a electrical charge
running through it with the help of a powers ourcetocarry
DNA and protein through the gel
GEL DOCUMENTATION SYSTEM
● Aka gel doc, gel image system or gel imager
● Visualize gel images
● Document nucleic acids and protein in gels
● UV light and camera/image capturing
POLYMERASE CHAIN REACTION (PCR) MACHINES
● Aka Thermal cyclers or Thermocyclers
● Amplify DNA; highly specialized instruments
● Regulates temperature of the reaction
● 3 step reaction:
○ Denaturation
○ Annealing
○ Extension
● Application of PCR:
○ Genotyping, Cloning, Mutation detection, Sequencing,
Microarrays, Forensics, Paternity testing
CHROMATOGRAPHY EQUIPMENT
● Qualitative and quantitative analysis
● Column chromatography –most common for protein
purification
● Gas chromatography –simple, highly sensitive
M olecular Biology & Diagnostics M AKATAK 4
BASIC LABORATORY SKILLS enzyme mix required volume
https://www.youtube.com/watch?v=DJVNI1fgCN4 EXAMPLE:
PIPETTING EXERCISES ● Thereare53s amplestobetestedandyouneedtoprepare
● MICROPIPETTE:plungerisdepressedbythethumbandas foram asterm ixusingBGItestkit.Thereare3controlsyou
it is released, liquid is drawn into adisposableplastictip; need to use and to possible pipetting error is drop into2.
when the plunger is pressed again, the liquid is dispensed. What is the total volume of master mix you need to prepare?
PARTS OF THE MICROPIPETTE ● Get the overall total (N).
● Plunger button ● N = samples + control + error = 53 + 3 + 2
● Volume adjustment ● N = 58
● Tip ejector button
● Tip cone/attachment
● equired volume for reaction mix = RM x N
R
PROPER HANDLING OF MICROPIPETTE ● = 18.5 uL x 58
● = 1073 uL
● Make s ure it is placed in the right amountofvolumetobe
● Required volume for enzyme mix = EM x N
dispensed
● = 1.5 uL x 58
● Add the appropriate disposable pipette tip and press the
● = 87 uL
plunger on its first stop
● TOTAL MASTER MIX = required volume for RM + EM
● Insert the tip into the s olution to be transferred ands lowly
● = 1073 uL + 87 uL
aspirate
● TOTAL MASTER MIX = 1,160 uL
● Move the pipette tip and dispense to the desired tube by
pressing the plunger pass the first stop then to second stop
● ou will now aspirate 1,073 uL of RM and 87 uL EM in a
Y
● Eject the disposable tip
s ingle tube and mix.
COMMON ERRORS IN HANDLING PIPETTES
● Afterm ixing,aspirate20uLofyourm asterm ixanddispense
● Using uncalibrated micropipettes to 53 micropipette tubes.
● Using contaminated solution or contaminated tips
● Aspirating too quickly
PH METER
● Pipetting an angle can cause volume variation
● easure the hydrogen ion activity in a s olution indicating
M
REAGENT PREPARATION acidity of alkalinity
MASTER MIX ● pH of a s olution is the m ost commonly performed
● Premixedconcentrateds olutionthathasallthecomponents m easurement in the molecular laboratory
for a real time PCR reaction which includes DNA ● Buffers olutionpH7.0(+/-0.02)isusedasas tandardbuffer
polymerase, dNTPs, MgCl2 and buffer in molecular laboratory
MASTER MIX PREPARATION ● It is mainly used as a calibration buffer for pH meters
● PCR Reagent Preparation involves the creation of “master ● A buffer s olution isonewhichresistschangesinpHwhen
m ix” s mall quantities of an acid or an alkali are added to it
● In BGI test kit, the master mix consists of the reagents: ● It functions by m easuring the voltage developed between
● Reaction mix two electrodes immersed in the s ampleandcomparethat
● Probe value to a calibration derived fromthes ameelectrodepair
● Primers and known standards
● Free nucleotides (dNTPs) ● These s tandard buffer s olutions m ust be accurate and
● Enzyme mix reliable and are used for pH meter calibration
● Taq polymerase – a thermostable DNA polymerase PARTS OF THE pH METER
(Thermus aquaticus) ● Display screen
● Reverse transcriptase ● Selection keys
HOW TO COMPUTE FOR THE MASTER MIX? ● Electrode holder
1. Check the package insert and look for the protocol ● Probe
requirement. CALIBRATING PH METER
○ Reaction mix should be 18.5 uL per well (RM) ● TWO-POINT CALIBRATION
○ Enzyme mix should be 1.5 uL per well (EM) ○ When using acidic solution, use a buffer that is acidic
○ RM + EM = 20 uL per well ○ When using basic solution, use a buffer that is basic
2. Categorize w hat you need for the test and the possible PROCEDURE
errors that may occur during pipetting. 1. Rinse the electrode with distilled water.
○ Total number of samples to be tested 2. Place the electrode in a neutral buffer with pH 7.00
○ Total number of controls to be used s olution.
○ Total number of pipetting errors 3. Waitforthem etertos tabilizeandadjustthem eteruntilit
○ Overall total = N reads a pH of 7.00.
3. Get the required volume of the RM & EM. 4. Removetheelectrodefromthes olutionandrinseitwith
○ Required volume of reaction mix = N x RM distilled water.
○ Required volume of enzyme mix = N x EM 5. Place the electrode in the s econd buffer that is either
4. Get the total volume of your master mix. acidic or alkaline.
○ Master m ix (uL) = reaction m ix required volume + 6. Waitforam omenttos tabilizethenadjustthem eteruntil
M olecular Biology & Diagnostics M AKATAK 5
it displays the s ame pH as thebufferyouhaveusedto
c alibrate.
. Re-place the electrode in the pH 7.00 buffers olution.If
7
the reading does not return to 7.00pH then repeat the
calibration procedure using both buffers.
M olecular Biology & Diagnostics M AKATAK 6
ISOLATION TECHNIQUES
https://www.youtube.com/watch?v=matNxUzRwUU B. DIFFERENTIAL EXTRACTION
DNA ISOLATION ● Specimen: sperm, epithelial cell, blood
● DNAcanbeisolatedfromtwodifferenttypesofcellswithout
SPECIMEN FOR DNA ISOLATION AND ITS YIELD
m ixing their contents
DNA YIELD (w ithout ● Modifiedtechniquefors electiveisolationofDNAforprofiling
DNA YIELD (w ithamplification)
amplification) in cases of sexual assaults
0.5ml serum or Procedure
1 ml blood 10-50ug 0.3-3ug
plasma 1. Epithelial cells are s electively lysed by the addition of
0.5cm dried SDS and Proteinase K
1ml buffy coat 50-200ug 0.04-0.7ug
blood 2. Centrifugation is used to s electively pellet any intact
1ml bone s perm cells
100-500ug 1ml saliva 5-15 ug
m arrow 3. The s upernatant with both m ale and female epithelial
107 cultured 500mg bone or cells can be removed to a separate tube
30-70 ug 30-50ug
cells teeth 4. Inthefirsttube,theisolateds permcellsarelysedbythe
1mg solid 1 pc hair addition of SDS, proteinase K and dithiothreitol to
1-10ug 0.1-0.2ug releasethes permcellandonthes econdtube,thelysed
tissue follicles
0.5ml bacterial epithelial cells are kept
10-35 ug 1mg feces 2-100pg 5. Purifybothtubesbyeitherorganicornon-organicm ethod
culture
1mg feces 2-228ug and transfer to a new set of tubes
C. HOMOGENIZATION
MATERIALS
● Specimen: fresh tissue or frozen tissue
● uffer agent
B
● Processofdisruptingbiologicalm embranesbringingtissue
○ TE buffer contains 10mM Tris and 1mM EDTA at pH
orcells ampletoahomogenousoruniforms tates uchthat
7.5-8.5
all fractions are identical in composition
● Additives
○ Mechanical method
○ NaCl: prevents DNA denaturation ○ Chemical method
○ Detergent: causes the cell to lyse m aking DNA ● Rotor-stator: contains a blade and perforated tube which
released in the solution rotates at high speed
● Others Procedure
○ EDTA:chelatingagentsthatblockstheactivityofDNase 1. Weigh all the tissue sample
enzyme 2. Grind or homogenize the tissue
○ MgCl2: protects DNA from mixing with other organelles 3. Centrifuge the sample at 10,000rpm for 10 minutes at 4C
○ Alcohol: allows precipitation of DNA 4. Collect the homogenate or the s upernatant into a
○ Proteinase K: digests contaminating proteins pre-chilled tube
○ Dithiothreitol: s tabilizes enzymes that possess free
s ulfhydryl groups D. MICROORGANISM
○ TritonX-100:lysingbacterialcellstoextractproteinand ● Somebacteriaandfungihavetoughcellwallsandm ustbe
other organelles broken down to release the nucleic acid and isolate the DNA
SAMPLE PREPARATION ○ Mechanical Method
A. DENSITY GRADIENT CENTRIFUGATION ○ Chemical Method
● Specimen:bloodinheparinwithphosphatebuffers alineor ■ 1% s odium dodecyl s ulfate + 0.2M NaOH +
acid citrate dextrose Tris-base, EDTA and glucose can break bacterial
● Based on s ize and density by employing a m edium of cell walls
graded densities that can s eparate peripheral blood ■ 8%s ucrose,8%tritonX-100,Tris-buffer,EDTAand
m ononuclear cell from other blood cells lysozyme treatment can release DNA immediately
● Ficoll-Paque: m ost common used gradient m edia for but should be precipitated with alcohol immediately
isolating mononuclear cells
○ Slow or s hort centrifugation, particles s eparate based ISOLATION METHODS
on size A. ORGANIC ISOLATION METHODS
○ Longorfastcentrifugation,particless eparatebasedon ● Hydrophobic contaminants s uch as lipids andlipoproteins
density s hould be dissolved
Procedure ● Uses high s alt, low pH and organic m ixtureofphenoland
1. Dilute blood sample to a 1:1 volume ratio chloroform for purification
2. Add 1 ml of density gradient medium to a fresh tube ● Phenol and chloroform form a biphasic emulsion when
3. Layer the diluted blood on top of the density gradient centrifuged; DNA will dissolve and isolated in the upper
4. Centrifuge at 400g for 30 minutes with brake off aqueous phase of the solution
5. After centrifugation, carefully harvest the m ononuclear ● Cetyltrimethylammonium bromide (CTAB): used as
cells on top of the gradient at the plasma layer of the tube pre-treatmentfors mallamountsofDNAfroms ampleswith
6. Wash the harvested cells twice in the appropriate buffer s mall quantity of DNA samples
M olecular Biology & Diagnostics M AKATAK 7
● RNase: enzyme used to degrade RNA contaminants
B. INORGANIC ISOLATION METHODS
● Also known as the salting out method
● Uses low pH, high s alt condition to precipitate proteins
leaving DNA in the solution
● DNAcanalsobeprecipitatedbyusingisopropanolpelleted
and resuspend in TE buffer
C. SOLID PHASE ISOLATION
● More rapid and comparable effective DNA extraction
● Can be performed using s olid m atrices to bind and wash
DNA
● Uses silica-based products such as diatomaceous earth
● Ideal for the viral and bacterial DNA isolation
NA isolation
R
SAMPLE PREPARATION
A. GUANIDINIUM THIOCYANATE PHENOL-CHLOROFORM
EXTRACTION
● Widely used method for extraction
● Guanidinium thiocyanate is a chaotropic agent for protein
degradation
B. SPIN COLUMN METHOD
● Alternative m ethod to guanidium m ethod; faster and less
toxic
● Samples should be homogenized before isolation
C. POLY(A) ISOLATION
● Poly(A) is the template for protein translation and m ost
m RNA carry this
● 1-2% of total RNA is made up of mRNA
● Separated by affinity chromatography on oligo(dT) cellulose
M olecular Biology & Diagnostics M AKATAK 8
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