Documenti di Didattica
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2. Autosomal recessive inheritance
Ex: Cystic fibrosis, Phenylketonuria
2
4. X-linked dominant inheritance
Ex: Incontinentia pigmenti, Vitamin-D resistant rickets.
Mitochondrial inheritance
Ex. Diabetes Mellitus with deafness
3
Deviation in the patterns of classical Inheritance patterns
1. Uniparental disomy:
It is defined as the presence of a disomic cell line containing two
chromosomes of a given kind inherited from one parent. Ex.. a female with
cystic fibrosis was found to have two identical copies of most or all of her
maternal chromosome 7.
2. Pseudodominant pedigree patterns:
In case a recessive character is common in the population then the
pedigree pattern resembles the dominant inheritance pattern.
3. Genomic imprinting:
In a considerable number of genetic disorders the expression of the
disease phenotype depends on whether it has been inherited from father
or from mother. This is called genomic imprinting. Ex.Prader willi and
Angelman syndrome.
4. Non- Penetrance:
Some times an occasional skipping of generation is observed in dominant
conditions. A person carrying a mutant gene fails to manifest the disease
but produces affected offspring. This is known as non-penetrance.
5. Anticipation:
A condition in which an inherited disease displays increasing severity
and/or an earlier age of onset in subsequent generations (dynamic
mutation). Ex. Huntington’s disease.
6. Mosaicism
Existence of more than one genetically distinct cell line following a single
fertilization event. Ex. XO / XX.
7. Chimerism
Existence of different cell lines from multiple fertilizations. Ex. XX / XY.
4
Fig 1.The common symbols used in drawing pedigrees
5
Cytogenetic Laboratory Set up
The sterile working place is the most important and basic requirement in the cell
culture laboratories.
The sterility should be maintained in the culture room and cleaned
with antiseptic solutions before starting culture work.
Room and laminar hood should be provided with an ultraviolet light
(30 watts), which should be switched on for an hour prior to use. UV light
should be switched off and laminar flow should be on while working in the
hood.
Entry to the culture room should be restricted. Persons using
culture room should wear an autoclaved gown or at least a clean apron.
Contamination of media, sera, phytohaemagglutinin is the greatest
hazard in culture procedure. Care must be taken in the selection and
preparation of materials to be used for tissue culture.
Medium should be prepared under sterile condition under laminar
flow. All glassware/water used should be sterilie.
Table tops and laminar hood should be cleaned with spirit.
Culture room should be steamed or fumigated at least once a
month and must be maintained dust free.
Equipment
Laminar hood
CO2 Incubator
Media filter assembly
Hot air oven
Preparation of Glassware
Prepare 30% acid solution with concentrated HCl or H2SO4 to soak the glassware
overnight. Clean the glassware and transfer to soap water and leave them
overnight. Care must be taken that they are completely dipped in soap or acid
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solution. With the help of bottle brush, scrub the glassware thoroughly both inside
and outside. Rinse several times under running tap water and then rinse with
distilled water thrice and dry in a hot air oven.
Apparatus
Filtration assembly with vaccum pump, conical flasks, disposable syringes
and needles.
Prepare media in the culture room using all aseptic precautions. Weigh 10 gms
of RPMI – 1640 nutrient medium and dissolve in one litre of autoclaved triple
distilled water. Add 0.5 ml of streptomycin and 0.5 ml of gentamycin and add
sodium bicarbonate (NaHCO3) and adjust pH 7.0 with 1N HCl or 1N NaOH. Store
the medium at 40C. Filter the media through millipore membrane of 0.22µm and
store it at 40C.
7
Preparation of peripheral blood cells for chromosome analysis
8
Preparation of stock medium
RPMI 1640 Lyophilized - l0 g
Triple distilled water - 100 ml
Colchicine solution
a) Stock solution
Colchicine powder - 6 mg
Triple distilled water - 10 ml
b) Working solution
Colchicine stock solution - 0.6 ml
Triple distilled water - 9.4 ml
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c) Hypotonic solution (O.O75M KCl)
Potassium chloride - 560 mg
Triple distilled water - 100 ml
e) Carnoy's fixative
Methanol and acetic acid in a ratio of 3: I.
f) Giemsa stain
i. Stock solution
Giemsa powder -1g
Glycerol - 66 ml
Methanol - 66 ml
To one gram of Giemsa powder 66 ml glycerol is added, gently mixed and
warmed at 600C in a water bath for one hour and cooled to room temperature.
Then 66 ml methanol is added and kept in the refrigerator for fifteen days. It is
then filtered and used as stock.
Working solution
Giemsa stock solution - 2 ml
Phosphate buffer - 2 ml
Triple distilled water - 46 ml
ii. Phosphate buffer ( 0.025M KH2PO4 )
Potassium dihydrogen
ortho-phosphate - 3.4 g
Triple distilled water - 1000 ml (pH 6.8 )
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Culturing procedure
1. Initiation
1ml of whole blood is obtained after venipuncture into a heparinized
syringe. 0.5ml of the whole blood is added to 5 ml of the working culture
media in duplicates. Cultures are then incubated at 37°C for 72 hours in a
CO2 incubator.
2. Harvesting
Harvesting is carried out by arresting the cells at metaphase by
adding 0.2ml of working colchicine solution at 70th hour, shaken well and
incubated for another two hours. The cultures are then harvested at 72nd
hour as follows:
a) The cultures are gently shaken and transferred to clean
centrifuge tubes and centrifuged at 1500 rpm for 10 minutes.
b) The supernatant is discarded and 5 ml of pre-warmed hypotonic
solution is added to the cell pellet and mixed well. The cell suspension is
incubated for 20 minutes at 37°C. Hypotonic solution is used to swell the
cells for better spreading of the chromosomes.
c) Cell suspension is centrifuged at 1500 rpm for 10 minutes and
the supernatant is discarded. The cells are then fixed in 5ml of freshly
prepared Carnoy's fixative. The cells are kept in the refrigerator overnight
after the first wash with fixative.
d) Later, 3 to 5 washes are given until the cell pellet is clear.
Slide preparation
a) After the final wash, the cells are suspended in 0.5 ml of fresh fixative.
Few drops of the cell suspension are then dropped on to clean pre-chilled
slides.
b) Slides are heat dried at 20°C on a hot plate for applying banding
techniques. Prepared slides are then stored until further use. Slides are
subjected to different staining procedures as follows:
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Application of Giemsa (G) banding
G banding is carried out by a modified method of Seabright (1971).
Chemicals required
Sodium chloride, trypsin, citric acid, sodium phosphate dibasic
anhydrous, methanol and giemsa.
Preparation of solutions
1) Trypsin solution
Stock solution
Trypsin - 25 mg
Physiological saline - 10 ml
Working solution
Trypsin stock solution (0.25%) - 1 ml
Physiological saline - 49 ml
4) Staining solution
Na2HPO4 - 8 ml
Citric acid - 1.5 ml
Methanol - 1.5 ml
Giemsa - 1 ml
Triple distilled water - 38 ml
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Procedure
1. Mark the patient’s name, accession number and date on the frosted end of
the slide.
2. Slides should be aged at 600C - 650 C for 2days or at 900C for 1 hour in
the oven.
3. Cool slides to room temperature in covered box.
4. Immerse slides in Trypsin solution. The amount of time varies and should
be adjusted according to age of slide.
5. Immerse slides in Phosphate buffer Saline solution to stop the action of
the trypsin.
6. Place the slide in the coplin jar containing the Giemsa stain for 7 minutes.
The length of time in the stain may vary considerably.
7. Rinse the slides in deionized or distilled water. Wipe the back of the slides.
8. Allow slides to air dry for 10 minutes.
9. Observe under the microscope and look for the banded metaphase
spread for the chromosomal analysis.
Scoring Metaphases
A minimum of 20-25 intact spread metaphses are analysed with image
analyser using Leica software. International System for Human Chromosome
Nomenclature (ISCN 1978) is followed for the construction of karyotypes.
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A schematic representation of Cytogenetic Analysis for screening chromosomal defects.
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Normal female Karyotype showing 46 XX chromosome constitution
15
Down’s Syndrome showing 47 XY (trisomy 21) chromosome constitution
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Fluorescence In Situ Hybridization
FISH Probes
Centromeric Probes
Centromeric probes containing alphoid-satellite repetitive DNA sequences
are small insert size clones and are used to detect chromosome copy
number in a cell.
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Equipment / Materials required
Waterbath, shaker incubator, hot plate, microfuge tube,cleaned frosted
glass slides, coverslips (24X24), rubber cement, nail paint.
Reagents required
Sodium chloride, Trisodium citrate, Formamide, Ethanol, NP-40, 4,6,-
diamidino-2-phenylindole (DAPI), 1,4-diazabicyclo[2.2.2]octane (DABCO).
Preparation of reagents
2X SSC buffer
0.882 gm trisodium citrate and 1.753 gm sodium chloride dissolved in 100
ml of distilled water.
Formamide (70%)
35 ml of formamide in 15 ml of 2X SSC buffer
Ethanol series
70%,90% and 100%.
Post hybridization washing solution I
49 ml of distilled water and 1ml of 20X SSC buffer and 150µl of NP-40
(derivative of Tween 20) solution.
Post hybridization washing solution II
45 ml of distilled water and 5 ml of 20X SSC buffer and 50 µl of NP-40
solution.
DAPI stain (stock)
2mg/ml (2X SSC) of DAPI
DAPI stain (working)
10µ l of DAPI in 100ml of 2X SSC.
Antifade solution
0.233gm DABCO is dissolved in 800µl of distilled water and add 200µl of
1M Tris-HCl and 9ml of glycerol.
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Method (direct labeled probes)
Slide preparation
1. Metaphase preparations using standard lymphocyte cultures fixed in
carnoy’s fixative are dropped on cleaned frosted slides.
2. The slides are dehydrated with 70%, 90%, 100% ethanol series for 5
minutes in each. (if the cytoplasm is not cleared pepsin treatment is
recommended). The slides are air dried at room temperature.
3. Keep the slides in 70% formamide at 730C in waterbath for 5 min and
denature the metaphases /interphase cells.
4. Transfer the slides into cold ethanol series of 70%, 90%, 100% for 5 min
each.
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FISH analysis
The slides are analysed with fluorescence microscope fitted with
CCD camera.
The microscope should have triple band filters for FITC, CY3 or
Texas red or Rhodamine, and DAPI.
Score non-overlapping individual cells or metaphases with intense
bright signals using 10x and 100x magnification.
Do not count cells with overlapping signals in interphase cells.
Cells with diffuse or split signal is considered as one signal.
Use optimum combination of filter set according to fluorochromo
excitation of signal and counter stain.
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DNA Isolation
DNA can be isolated from all the nucleated cells such as hair, tissue,
blood etc. Certain sources contain high levels of proteins and many types of
secondary metabolites that affect DNA purification. Highly purified DNA is
essential for molecular studies.
Generally DNA is isolated from the human blood using the following two
methods based on classical principles of lysis and purification:
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Isolation of Genomic DNA
by Phenol Chloroform or Enzymatic Method
The most basic of all procedures in molecular biology is the isolation and
purification of nucleic acids. The key step, the removal of proteins, can often be
carried out simply by extracting aqueous solutions of nucleic acids with phenol,
chloroform and isoamyl alcohol. Additional measures are required when nucleic
acids are purified from complex mixtures of molecules such as cell lysates. In
these cases, it is usual to remove most of the proteins by digestion with
proteolytic enzymes such as proteinase k, which are active against a broad
spectrum of native proteins before extracting with organic solvents.
Equipment
Refrigerated centrifuge
Water bath
Reagents
2. 20% SDS
20grams of sodium dodecyl sulphate is dissolved in 100ml of distilled
water. It is an anionic detergent.
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3. Proteinase K
20mg of Proteinase K is dissolved in 1ml of autoclaved distilled water.
4. Phenol
Phenol is saturated with equal volume of Tris (pH 7.8) until a pH of 7.8 is
obtained. Liquified Phenol is a clear colourless liquid and can be used for
molecular work without reinstallation. Equilibrated phenol is available with
Banglore Genei.
5. 1 M Tris
121.1g of Tris base is dissolved in 800ml of water. The pH is adjusted to
the desired value by adding concentrated HCl. The pH of Tris solutions is
temperature dependent and decreases approximately by 0.03 pH for
every 1oC rise in temperature. The solution is allowed to cool in room
before making final adjustments to the pH. The volume is adjusted to 1
litre with water and sterilized by autoclaving.
6. Chloroform-Isoamylalcohol
Chloroform and Iso-amylalcohol are mixed in 24:1 ratio.
Iso-amylalcohol is frequently used to remove proteins from preparations of
nucleic acid. It also reduces foaming during extraction.
7. 3M Sodium Acetate
408 mg of Sodium acetate is dissolved in 100ml distilled water.
8. Absolute Ethanol
100% Alcohol serves as Absolute Ethanol
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9. 70% Ethanol
70ml of Ethanol is made upto 100ml with distilled water
PROCEDURE
1. To 5ml of heparinized whole blood add three times equal volume of
erythrocyte lysis buffer. Shake gently and keep it in ice for 15 minutes.
2. Remove tubes from ice and centrifuge in a refrigerated centrifuge at 3,500
rpm for 10 minutes at 4°C.
3. Discard supernatant and disturb the pellet with 1 ml of erythrocyte lysis
buffer and make up to 5ml with buffer (repeat the step until white pellet is
obtained).
4. To the sample add 300 µl of 20% SDS and mix gently for 3 to 4 times. To
this mixture, add 40µl of Proteinase K and incubate at 37°C in a water
bath overnight.
5. After overnight incubation, add 5ml of phenol mix slowly and centrifuge at
10,000 rpm for 10 minutes.
6. Transfer the supernatant to a fresh autoclaved tube. To this, add 5ml of
Phenol: Chloroform-Isoamylalcohol (25:24:1). Mix gently and then
centrifuge at 10,000 rpm for 10 minutes at 4oC.
7. Transfer the supernatant to a fresh autoclaved tube and add 5 ml
chloroform and Isoamylalcohol (24:1). Mix and centrifuge at 10,000rpm
for 10minutes.
8. Transfer the supernatant to a fresh autoclaved tube and add 3 volumes of
chilled ethanol and keep overnight at -200C. Later centrifuge at 10000rpm
for 10 minutes at 40C.
9. Discard the supernatant and wash the pellet with 70% ethanol and allow it
to dry.
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10. After the pellet gets dried up, it is dissolved in 200µ l of TE Buffer and
transferred to 1.5ml eppendorf tube for storage.
Equilibration of Phenol
Before use, phenol must be equilibrated to a pH > 8 because the DNA partitions
into organic phase at acid pH. Store liquefied phenol at -20°C. As needed,
remove the phenol from the freezer and then melt it at 68°C. Add
hydroxyquinoline to a final concentration of 0.1%. This compound is an
antioxidant, a partial inhibitor or RNase and a weak chelator of metal ions.
To the melted phenol, add an equal volume of Tris buffer (0.5M Tris, pH 8). Stir
the mixture on a magnetic stirrer to 15min. Two phases are formed – upper
aqueous phase removed with the help of glass pipette attached to a vacuum line
equipped with appropriate traps.
Add equal volume of 0.1M Tris to the phenol. Stir the mixture on a magnetic
stirrer for 15 min. Remove the aqueous phase as described in step 2. Repeat
the extraction until the pH of the phenolic phase is >7.8 and store at 40C until
use.
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2. Isolation of Genomic DNA by “Salting Out” Technique
The salting out technique has been used successfully to isolate large quantities
of human DNA from whole blood. DNA is extracted from peripheral blood
leucocytes using 5ml of whole blood.
Equipment
Centrifuge
Microcentrifuge
Spectrophotometer
Electrophoresis unit
DC Power supply
Incubator
Reagents
1. TKM 1 buffer (RBC Lysis buffer) 500 ml
Tris HCl (10 mM) pH 7.6 - 0.605 g
KCl (10 mM) - 0.372 g
MgCl2 (10 mM) - 1.016 g
EDTA ( 2mM) - 0.372 g
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For the preparation of TKM1 and TKM 2 buffers the pH should be maintained by
dissolving Tris in about 70ml. of water and pH is adjusted to 7.6. Now dissolve
EDTA followed by the rest of the chemicals.
Procedure
1. 5 ml of blood is drawn in EDTA vaccutainer, mixed well by inverting the
tube and stored at 4oC till use.
2. Bring the blood sample to room temperature. Then transfer 5 ml of blood
into 15 ml centrifuge tube and add equal volume of TKM1 buffer.
3. Add 100 µl of NP-40 or Triton –X to lyse the red cells. Mix well by
inversion.
4. Centrifuge at 2200 rpm for 10 minutes at room temperature (RT) in a
Beckman table top centrifuge.
5. Slowly pour off the supernatant and save the nuclear pellet (the small
pellet settled at the bottom of the tube) and wash the pellet in 5 ml of
TKM1 buffer and centrifuge as before. Repeat the step if lysis is
incomplete.
6. Add 800µl of TKM2 to the pellet and resuspend the cells.
7. Add 125µl of 10% SDS to lyse the WBCs and mix the whole suspension
thoroughly and incubate for 10 min at 550C. Later transfer the contents
to sterile eppendroff tube.
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8. Add 300 µl of 6M NaCl to the tube and mix well to precipitate the proteins
by inversion and centrifuge at 12000 rpm for 5 min in a micro centrifuge.
9. Save the supernatant containing DNA and discard the pellet containing
precipitated protein.
10. Transfer the supernatant to a 15 ml tube and add 2 volumes of 100%
ethanol at room temperature. Invert the tube several times slowly till the
DNA precipitates.
11. Remove the precipitated DNA strands with a glass rod and transfer to a
fresh eppendroff tube containing 1 ml of ice cold 70% ethanol.
12. Centrifuge the sample for 5 min at 12000 rpm at 4o C.
13. Dry the pellet in speed vac or in an incubator and resuspend it in 500 µl of
Tris –EDTA buffer at 65oC for 15 min.
Pour off the supernatant to the pellet add 5ml of TKM1 buffer centrifuge as before
28
Add 125µl 10% SDS and incubate for 10min at 550C
Transfer the DNA to the fresh eppendorf tube containing 70% ethanol
Dry the pellet and resuspend DNA in 500µl of Tris EDTA buffer at 650C for 15min
Estimation of DNA
The isolated DNA needs to be studied for its quality & quantity by using the
following methods.
Spectrophotometric Method
Electrophoretic Method
Spectophotometric Method
1. It is an analytical method used for determining the purity (quality) &
quantity of the isolated DNA.
2. The absorbance is measured at 260nm, at this wavelength an absorbance
of 1.0 corresponds to 50 µg per ml of dsDNA, 40µg/ml of ssDNA or RNA &
20µg/ml of oligo nucleotides.
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3. The ratio of absorbances at 260nm & 280nm (O.D260/280) provides an
estimation of the purity.
4. For pure DNA and RNA the ratio is approximately 1.8 and 2.0
respectively.
5. If DNA is contaminated with proteins then the ratio will be < 1.8
6. If DNA is contaminated with RNA then the ratio will be > 2.0
Equipment
Spectrophotometer
Reagents
1. DNA sample
2. T.E Buffer
10mM Tris HCl (pH 7.5)
1mM EDTA (pH 8.0)
3. Sterile distilled water
Procedure
1. Add 10µl of isolated DNA sample to 2490µl of T.E buffer / sterile
distilled water (250 fold dilution)
2. Mix well & measure the absorbance at 260nm and 280nm in the
spectrophotometer to determine the quality & quantity of DNA.
3. Calculate the concentration of DNA in the sample by the following
formula:
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Calculate the ratio of absorbance at 260nm and 280nm
- If DNA is pure then the ratio will be 1.8-2.0
- If DNA is contaminated with Proteins then the ratio will be <1.8
- If DNA is contaminated with RNA then the ratio will be >2.0
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Agarose Gel Electrophoresis
The agarose gel electrophoresis is an analytical method basically used for
purification of DNA fragments. Agarose which is extracted from seaweeds, is a
linear polymer of alternating D and L galactose, which are linked by alpha (1-3)
and beta (1-4) linkages. The basic structure is
AGAROSE
Agarose gels are cast by melting the agarose in the presence of the
desired buffer until a clear, transparent solution is achieved. The melted solution
is then poured into a mold and allowed to harden.
Principle
DNA molecules are negatively charged, under electrical field they migrate
through the gel depending on size towards the positively charged electrode and
thus DNA bands can be separated. Migration of DNA molecule through the pores
of the matrix plays an important role in molecular weight separation. The relative
mobility depends upon the concentration and the type of agarose used, the
strength of the applied current, the ionic strength and the density of the DNA
bands separated. Larger molecules move more slowly because of greater
frictional drag and because they worm their drag through the pores of the gel less
efficiently than smaller molecules. Thin gels yield dramatically better results than
thick gels; so cast gels only thick enough to contain the volume of DNA that will
be loaded
Equipment
Gel Electrophoresis unit, moulds, combs, power pack
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Reagents
10X Tris Borate Buffer (pH 8)
109g Tris
55.6g boric acid
9.3g EDTA
Make up the volume to 1 liter with distilled water.
Procedure
1. Prepare 1X TBE by diluting 10X TBE with double distilled
water.
2. Take 40ml of 1X TBE in a 200ml conical flask and add
600mg of agarose. Boil to dissolve agarose and allow it to cool.
3. Mean while, adjust the combs in electrophoresis set in such
a way that comb on the side is about 2cm from the cathode.
4. When the temperature is around 450C add 2µl of ethidium
bromide to visualise DNA under UV gel doc.
5. Pour the solution slowly into gel tank without creating
bubbles.
6. Once the gel gets solidified place the electrophoresis set into
the tank and pour 1X TBE buffer till the level stands 0.5 - 0.8cm above gel
surface.
7. Add 5µl of isolated genomic DNA on 2% agarose gel along
with the DNA known amount (Marker DNA) mixed with loading dye.
8. Connect the cathode and anode cords to the power pack,
and adjust the voltage to 60V and run till the dye reaches ¾ of the gel.
9. Then visualize the DNA under the UV gel doc
10. The concentrations of DNA in the unknown sample can be
obtained by comparing the band intensities with the marker DNA.
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Polyacrylamide Gel Electrophoresis
ACRYLAMIDE
34
The distinct advantage of acrylamide gel systems is that the initial concentrations
of acrylamide and BIS control the hardness and degree of cross linking of the
gel. The hardness of a gel in turn controls the friction that macromolecules
experience as they move through the gel in an electric field, and therefore affects
the resolution of the components to be separated. Hard gels (12-20%
acrylamide) retard the migration of large molecules more than they do small
ones. In certain cases, high concentration acrylamide gels are so tight that they
exclude large molecules from entering the gel but allow the migration and
resolution of low molecular weight components of a complex mixture.
Alternatively, in a loose gel (4-8% acrylamide), high molecular weight molecules
migrate much farther down the gel and, in some instances, can move right out of
the matrix.
Note: Fragments differing by only few base pairs can be separated better by
polyacrylamide gel.
Equipment
Vertical gel electrophoresis system (Bangalore Genei Pvt.Ltd)
1mm thick combs and spacers
Power pack (capable of maintaining constant voltage of up to 400V)
Reagents
Preparation of 10 % non denaturing Polyacrylamide Gel (29:1)
Acrylamide Solution (30%) - 20 ml
10XTBE - 6 ml
Distilled water - 34 ml
APS (10%) - 400 µl
TEMED - 51 µl
Total volume - 60 ml
35
Preparation of Polyacrylamide Gel Slab
36
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is an enzyme catalyzed biochemical reaction in
which small amount of a specific DNA fragment is amplified into large amount of
linear double strand DNA using gene specific oligonucleotide primers. PCR is
commonly used for the detection of hereditary diseases, identification of genetic
fingerprints, diagnosis of infectious diseases, cloning of genes, paternity testing,
and DNA computing etc.
Principle
The DNA is amplified by selecting specific primers. Primers are short oligo
nucleotides usually 18-25 bp that are complementary to the beginning and end of
the DNA fragment to be amplified. They anneal (adhere) to the DNA template at
these starting and ending points, where the DNA-polymerase binds and begins
the synthesis of the new DNA strand using deoxynucleotide phosphates
(dNTPs). The PCR reaction is carried out in a thermal cycler. The PCR process
consists of a series of twenty to thirty-five cycles. Each cycle consists of three
steps.
(1) Denaturation
The double -stranded DNA to be heated to 94-96°C in order to separate
the hydrogen bonds of DNA helix. Prior to the first cycle, the DNA is often
denatured for an extended time to ensure that both the template DNA and
the primers have completely separated and is now single-strand only. The
appropriate time for the reaction is 1 to 5 minutes.
(2) Annealing
After separating the DNA strands, the temperature is lowered so the
primers can attach themselves to the single DNA strands. The
temperature of this stage depends on the primers and is usually 5°C
below their melting temperature (45-60°C). The appropriate time for the
reaction is 1-2 minutes. The melting temperature of a specific oligo
nucleotide primer (Tm) can be calculated by the following simple equation
37
Finally, the DNA-Polymerase has to copy the DNA strands using deoxy
nucleotide tri phosphates (dNTPs). It starts at the annealed primer and works
its way along the DNA strand. The extension temperature depends on the
DNA-Polymerase. The time for this step depends both on the DNA-
Polymerase and on the length of the DNA fragment to be amplified. As a rule-
of-thumb, minute per 1 kbp. A final extension step is frequently used after the
last cycle to ensure that any remaining single stranded DNA is completely
copied. This differs from all other extension steps, only in that it is longer,
typically 10-15 minutes.
Equipment
Thermal cycler
Reagents
The components required are:
• DNA template, or cDNA which contains the region of the DNA
fragment to be amplified Two primers, which determine the beginning
and end of the region to be amplified.
• Taq polymerase, which copies the region to be amplified.
• Nucleotides, from which the DNA-Polymerase builds the new DNA.
• Buffer, which provides a suitable chemical environment for the DNA-
Polymerase.
Procedure
To carry out a PCR experiment, the targeted DNA is mixed with a pair of gene
specific oligonucleotide primers, deoxynucleotides and Taq DNA polymerase.
38
PCR reaction mixture (50 µl) consists of the following components
Component Addition order Volume Final concentration
Sterile Distilled Water 1 35.5 µl
10X Reaction Buffer 2 5.0 µl 1X
dNTPs 3 2.0 µl 200 micro moles
Primer1 4 2.0 µl 20 pico moles
Primer2 5 2.0 µl 20 pico moles
Taq DNA Polymerase 6 0.5 µl 3 units/µl
DNA sample 7 3.0 µl 100 ng
Total mix 50 µl
All the tubes containing 50µl reaction mixture are placed in the thermal cycler.
The cycling conditions of PCR consist of the following steps:
39
Amplification of Refractory Mutation System –
Polymerase Chain Reaction ( ARMS – PCR )
Principle
A typical ARMS assay comprises two PCR’s, each conducted using the same
template DNA. Both reactions contain a common primer that anneals to an
invariant DNA sequence to one side of the mutation to be detected. The 3’
terminus of the common primer is oriented towards the mutation. In one of the
reactions, the second primer specific for one allele and in the other reaction the
primer is specific for alternative allele.
Primers are designed in such a way that 3’end residue is specific to the mutation
or the normal gene sequence, thus allowing the amplification to occur only when
primer sequence is complementary to the genomic DNA. Genomic DNA is
amplified by using two sets of primers, one internal control set and other specific
for normal / mutant gene.
Equipment
Thermal cycler
Horizontal electrophoretic unit with power pack
40
Reagents
Assay volume - 25µl
Genomic DNA - 0.5 µg
Primers - 0.2µM (each)
dNTPs - 30µM (each)
Taq polymerase - 0.5U
PCR buffer - 1X
Tris HCl - 10mM pH 8.4
KCl - 50mM
MgCl2 - 1.5mM
41
Amplification for Detection of HbS and HbE Mutations
Primers P7 & P8 along with P14 common primer is used for amplification of HbS
mutation. Primer P9 and P10 along with P14 common primer is used for HbE
mutation. Sample digested with Bhc II or Mnl I.
42
Multiplex PCR (M-PCR)
Multiplex PCR with its simplicity, reliability and cost effectiveness has gained
wider acceptability as a mutation detection method for the diagnosis of DMD and
for identification and distribution frequency of deletions. The deletion information
of the affected individual is necessary for carrier detection. Four sets of primers
which amplify 4 different exons in the DMD gene are used for the identification of
mutations in DMD patients.
Principle
The use of multiple, unique primer sets within a single PCR reaction to produce
amplicons of varying sizes specific to different DNA sequences. By targeting
multiple genes at once, additional information may be gained from a single test
run that otherwise would require several times the reagents and more time to
perform. Annealing temperatures for each of the primer sets must be optimized
to work correctly within a single reaction, and amplicon sizes, i.e., their base pair
length, should be different enough to form distinct bands when visualized by gel
electrophoresis.
43
PCR set up
44
Molecular Diagnosis of Haemoglobinopathies
by Reverse Dot Blot Technique
Principle
45
The following stepwise approach based on PCR and subsequent analyses of the
amplified product can be adopted for molecular characterization and prenatal
diagnosis of the thalassemia syndromes in Indian population.
46
9. Air dry the membrane thoroughly.
10. Incubate the membrane for 8 min in 0.1 N NaOH.
11. Wash the membrane thoroughly with DDW.
12. Air dry the membrane thoroughly.
Part - 2. Hybridization and colour development
1. Incubate the blotted membrane in hybridization buffer (2xSSC &
0.1% SDS) for 1/2. hr at 450C
2. Dilute PCR product with 30 µ l of hybridization buffer and denature
the sample at 95OC for 10min.
3. Add the PCR product to pre-hybridised membrane.
4. Continue hybridization for 1 hr at 450C.
5. In a tray / falcon tube add 50 ml hybridization buffer and incubate at
450C.
6. Take out all the strips from the 15 ml falcons & give a gentle wash
with hybridization buffer in a tray.
7. Transfer all the strips to 50 ml falcon containing hybridization buffer
and washing continued for 15 mins at 45oC
8. Take 10 ml hybridization solution in a tube; add 3.5 µ l streptavidin
labeled ALP conjugate. This step to be carried out in dark .
9. Pour the solution in a tray and transfer all the strips into the tray
10. Incubate at RT for 1 hr in dark
11. Transfer all the strips into another tray and wash with hybridization
buffer 3 times at intervals of 5 min each
12. Take 12 ml of Buffer C (50 mM Mgcl2, 100 mM Tris pH 9.5, 100 mM
NaCl)
13. Add 200 µ l substrate solution (NBT /BCIP). This step to be carried
out in dark.
14. Transfer all the strips into a tray and add the substrate prepared
Wait for colour development.
47
Restriction Fragment Length Polymorphism (RFLP)
RFLP is widely used technique to detect known mutations and variations using
specific restriction endonucleases. Genetic defects can be diagnosed by RFLP,
as normal and defective genes give rise to different restriction patterns, if change
is in the recognition site of restriction enzyme.
Principle
48
Procedure
1. Prepare reaction mixture by adding the following components.
PCR product - 10µl
Autoclaved distilled water - 6µl
10X assay buffer - 2µl
Taq 1 (2 units) - 2µl
Total 20 µl
2. Mix contents of each tube carefully and spin at full speed in a
microfuge for 1 minute.
3. Incubate the mixtures for 2 hours at 650C for digestion of PCR
products.
4. Load the samples in 3% agarose gel and electrophorese for two hours
at 60volts.
5. After electrophoresis stain the gel with ethidium bromide.
6. View the gel under UV gel documentation.
Interpretation
49
Single Stranded Conformation Polymorphism (SSCP)
Principle
Single stranded DNA has a tendency to fold and form secondary conformation
due to intra molecular bonds at sites of base complementation. These molecules
migrate in non denaturing gels depending not only on the size but also on the
conformation they take up. Any change in the nucleotide sequence affects the
conformation of the molecule which can be detected by shift in the mobility of the
bands on gels.
In this technique PCR amplified products are denatured and snap cooled before
loading onto non-denaturing gel. The electrophoretic mobility of ssDNA depends
on the configuration or shape adopted which is highly variable and sequence
dependent. The detection of sequence variation in DNA is important for the
identification of disease causing mutations in several genetic disorders.
Equipment
Vertical gel electrophoresis system
1mm thick comb and spacers
Power pack (should be capable of maintaining constant voltage of up to
400V)
50
Reagents
1. 10X TBE (pH 8.0)
Tris - 108g
Boric Acid - 54g
0.5M EDTA (pH 8.0) - 40ml
Adjust the pH and makeup the final volume to 1000ml with double distilled
water.
Procedure
1. Preparation of 10 % non denaturing Polyacrylamide Gel (29:1)
Acrylamide Solution (30%) - 20 ml
10XTBE - 6 ml
Distilled water - 34 ml
APS (10%) - 400 µl
TEMED - 51 µl
Total volume - 60 ml
Mix the reagents as specified above and pour into the vertical gel mold avoiding
air bubbles. Leave the mold undisturbed for 2 hours for polymerization. After
polymerization is complete, the gel plate is fixed to the unit. Fill the upper and
lower tanks of electrophoresis unit with 1X TBE buffer and pre run the gel for
30minutes at 100V to equilibrate the gel and to maintain the constant
temperature.
51
2. Denaturation of PCR products
Add 5µl of PCR product, 10µl of 95% Formamide and 2µl of loading dye
(0.05% Bromophenol blue; 0.05% xylene cyanol) into 0.2 ml PCR tubes,
mix well and denature at 950C for 5-10 min. Snap freeze in ice to prevent
re-annealing of denatured PCR products.
3. Load 10 µl of denatured samples into the wells of non-denaturing
polyacrylamide gel.
4. Carryout electrophoresis at 200V for about 12-14hrs. The temperature should
be maintained constant throughout the experiment.
5. Visualize the bands by silver staining.
Silver Staining
1. After electrophoresis, separate glass plates carefully to detach the gel and
place the gel in a plastic tray containing distilled water and gently rinse the
gel to remove the buffer.
2. Fix the gel in 10% ethanol by gently shaking for 10 mins.
3. Remove the ethanol by suction.
4. Add 0.7% of nitric acid just enough to cover the gel and gently shake the
tray for 6 mins.
5. Remove nitric acid by suction, add 0.02% of hypo and gently shake for
1min.
6. Rinse the gel in distilled water for 20 sec.
7. Add 0.1% silver nitrate and place in dark for 15mins.
8. Rinse the gel in distilled water for 20 secs.
9. Add 100 ml of developer (Hypo, Na2CO3, HCHO) to the tray.
10. Cover with aluminium foil and shake till the bands develop.
11. To avoid over developing of the gel add 5ml of 10% acetic acid or citric
acid to stop the reaction.
12. Store the gel in distilled water till the documentation.
52
Interpretation
The silver nitrate stained bands are examined for mobility shift in comparison to
control samples. Shift in mobility indicates presence of mutation in the target
region. When 3 or 4 bands are present, it indicates heterozygosity. Exact nature
of mutation can be confirmed further by sequencing.
53
High Performance Liquid Chromatography (HPLC)
Principle
HPLC is used to separate components of a mixture by using a variety of
chemical interactions between the substance being analyzed (analyte) and the
chromatography column.
54
column chromatography, alkali denaturation etc. High performance liquid
chromatography (HPLC) which is a relatively fast method, has been used for the
determination of various hemoglobins including A2 and F. The VARIANT HPLC
system is fully automated which can be used to separate and determine area
percentage for hemoglobin A2 and F and to provide qualitative determination of
abnormal hemoglobins. The most commonly occurring hemoglobin variants
include hemoglobin D, S, C and E.
Procedure
A. Preparation of Reagent
1. Elution buffers- readily available with kit.
2. HbA2/F calibrator
Reconstitute the calibrator vial by adding 10ml calibration diluent.
Swirl gently to dissolve and ensure complete mixing. Allow to stand
at room temperature for 10 minutes. Reconstituted calibrator is
stable for 10days at 2-8OC.
55
3. Whole Blood Primer
Prepare the whole blood primer by adding 10ml of de-ionized water
to the vial. Swirl gently to dissolve completely. Allow to stand for 10
minutes at room temperature. Reconstituted whole blood primer will
be stable for 21 days at 2-8OC.
B. Preparation of Sample
Whole blood is collected in a tube with EDTA as anticoagulant. The
samples are stable for 7 days when stored at 2-8OC.
Pipette 5µ l whole blood to 1.5ml sample vials.
Add 1ml of hemolysis reagent to each sample
Cover each sample vial with parafilm and mix by inversion.
Place the sample vial into VARIANT. Hemolysate is stable for 24
hours when stored at 2-8OC.
C. Run Setup
Prepare patient samples as mentioned before.
Transfer 250µ l each of primer, de-ionised water and calibrator into
3 sample vials.
Place the reagents and prepared hemolysates into the VARIANT
sample tray as shown below.
STAT Well Hemoglobin primer
Position 1 Deionized water
Position 2 Hb A2 / F calibrator
Position 3-100 Patient hemolysates
• Switch on the machine 20 minutes before the procedure. The
system comes to IDLE Status. Then only the procedure can be
started. Enter the programme and IDENTITY code of the samples.
Then START the procedure. Printed Chromatogram will come out
after 6.5 minutes.
56
• After completion of the run the system automatically initiates a
WASH cycle for 5 minutes. Then the system enters IDLE status
and the machine can be switched off.
D. Interpretation of Results
The chromatogram can be analysed based on the expected value range.
Patient state HbA2 HbF Level HbS Level HbD Level HbE Level
Level
Heterozygous 4 to 9% 1 to 5% _ _ _
β -Thal
Homozygous Normal or 80-100% _ _ _
β -Thal Increased
Heterozygous < 1.5% 10-20% _ _ _
HPFH
Homozygous Absent 100% _ _ _
HPFH
Heterozygous Normal Normal Increased
Sickle Hbs
Thus the VARIANT HPLC System is the easiest method to detect the carrier
status in hemoglobinopathies.
57
Newborn Screening by ELISA
Newborn Screening is basically the practice of testing every newborn for certain
harmful or potentially fatal disorder which is not apparent otherwise at birth. Many
of these are metabolic disorders, often called as inborn errors of Metabolism,
which interfere with the body’s use of nutrients to maintain healthy tissues and
produce energy. Other disorders that may be detected through screening include
problems with hormones, vitamin levels, or the blood. In general, metabolic and
other inherited disorders can hinder an infant’s normal physical and mental
development in a variety of ways. Also, parents can pass along the gene for a
certain disorder without even knowing that they are carriers.
With a simple blood test in which blood sample is taken by a heel prick from the
foot of the baby, doctors can tell whether newborns have certain conditions that
could eventually cause problems. Even though these conditions are considered
rare and most babies are given a clean bill of health, early diagnosis and proper
treatment can make the difference between lifelong impairment and healthy
development.
58
be detected immediately after birth and can be treated with oral doses of thyroid
hormone to attain normal development.
59
Prinicple
Reagents
1 Coated Reaction Wells Wells coated with monoclonal anti-TSH antibody
2 Conjugate Reagent Enzyme labelled anti-TSH antibody solution
(labelled with Horse-Raddish Peroxidase)
3 Substrate Reagent TMB
4 Stop Solution Reaction stop solution (1% sulphuric acid).
5 Wash Buffer 20 X Wash Buffer
Concentrate
6 Blood Spot Standards Whole Blood containing approximately 5, 10,
30 and 65 μ IU/ml. TSH spotted & dried on S & S
903 paper.
7 Blood Spot Controls Whole Blood containing approximately 3,7
& 30 μ IU/ml. TSH spotted & dried on S&S
903 paper.
8 Plate sealer plate sealing sheets
60
moisture–proof environment for up to 14 days. Care should be taken to assure
uniformity of blood spot punching. Spots should be punched from similar areas
and away from fingers and printed marks. Spots showing evidence of pooling,
clotting or incomplete saturation should be rejected. Specific rejection criteria for
questionable blood spots are the responsibility of the screening laboratory. Cord
blood should not be used as a specimen.
Note
Each laboratory should establish its own normal range and cutoff concentrations
to account for variations in patient populations. Values in the assay are
expressed in μIU/ml blood. The factor for converting μIU/ml whole blood to
μIU/ml serum is 2.22.
Procedure
The standards, controls and patient specimens are eluted directly in the coated
reaction wells with the Conjugate Reagent.
1. Punch 1 x ⅛ inch diameter disc from each standard and control or patient
specimen into a clean well of the coated reaction plate. Standards should
be punched in duplicate.
61
2. Add 100 μl of conjugate reagent into all wells containing standards,
controls and patient specimens.
3. Seal the wells. Place the micro titer plate on a shaker and shake at room
temperature (18 – 25˚C) for 3½ hours at 350 rpm. Ensure all punched
discs are immersed in the Conjugate Reagent throughout the shaking.
4. Decant the contents of the wells, including the punched discs into a
suitable container. Wash the plate with a plate washer, or manually, 5
times using a minimum of 300μl Wash Solution per well. After the last
wash, firmly tap the plate on absorbent paper to remove any residual
wash solution.
5. Add 100 μl of Substrate Reagent to all wells. Seal the wells with a Plate
Sealer and incubate the plate at room temperature (18 – 25˚C) for 30
minutes in dark.
6. Add 100 μl of Stop Solution to all wells. Gently mix the plate.
7. Read the plate at 450nm within 30 minutes.
Interpretation of Results
The recommended screening ranges are: Normal : 0 – 10 μIU/ml
Borderline : 10 - 20 μIU/ml
Abnormal : > 20 μIU/ml
62
Neonatal 17 - OHP Screening
Principle
Reagents
63
7 Blood Spot Standards Whole Human Blood containing approximately 0,
5, 10, 20, 50 and 100 ng/ml. 17 - OHP spotted &
dried on S&S 903 paper.
8 Blood Spot Controls Whole Human Blood containing approximately
7.5 & 40 ng/ml.
17 - OHP spotted & dried on S & S 903 paper
9 Plate sealer plate sealing sheets
• Wash Buffer
64
Prepare the wash buffer by mixing 1 bottle of 20X concentrated wash
buffer concentrate (100 mL) with 1900 mL of deionized water. The diluted
(1X) wash buffer is stable for 60 days at room temperature (18 – 25˚C).
Note
Each laboratory should establish its own normal range and cutoff concentrations
to account for variations in patient populations. Values in the assay are
expressed in ng/ml blood. The factor for converting μIU/ml whole blood to ng/ml
serum is 2.22. The factor for converting ng/ml whole blood to nmol/L whole blood
is: 0.33 ng/ml whole blood = 1 nmol/L whole blood
PROCEDURE
1. The standards, controls and patient specimens are eluted directly into
the coated wells with the conjugate Reagent.
2. Punch 1 x ⅛ inch diameter disc from each Standard and Control or
patient specimen into a clean well of the coated reaction plate.
Standards should be punched in duplicate.
3. Add 50 μl of conjugate reagent into all wells containing Standards,
Controls and patient specimens.
4. Add 50 μl of Antibody Solution to each well.
5. Seal the wells. Place the microtitre plate on a shaker and shake at
room temperature (18˚C – 25˚C) for 3½ hours at 350 rpm. Ensure all
punched discs are immersed in the Conjugate Reagent throughout the
shaking.
6. Decant the contents of the wells, including the punched discs into a
suitable container. Wash the plate with a plate washer, or manually, 5
times using a minimum of 300 μL Wash Solution per well. After the last
wash, firmly tap the plate on absorbent paper to remove any residual
wash solution.
65
7. Add 100 μl of Substrate Reagent to all wells. Seal the wells with a
Plate Sealer and incubate the plate at room temperature (18–25˚C) for
30 minutes in dark.
8. Add 100 μl of Stop Solution to all the wells and gently mix the plate.
9. Read the plate at 450nm within 30 minutes.
Interpretation of Results
The recommended screening ranges are : Normal : < 65 ng/ml
Borderline : 65 - 89 ng/ml
Abnormal : > 90 ng/ml
66
Comet Assay
The comet assay is a simple, rapid and sensitive technique for analysing and
quantifying DNA damage in individual mammalian cells. This was first introduced
by Ostling and Johanson in 1984. The image obtained looked like a “comet” with
a distinct head, comprising of intact DNA and a tail. The more versatile alkaline
method of the comet assay was developed by N.P. Singh and co-workers in
1988. This method was developed to measure low levels of strand breaks with
high sensitivity.
Principle
In the comet assay, the cells are embedded in a thin agarose gel on a
microscope slide. The cells are lysed to remove all cellular proteins, and the DNA
is subsequently allowed to unwind under alkaline conditions. Following unwinding
the DNA is electrophoresed and stained with silver nitrate. During
electrophoresis, broken DNA fragments (damaged DNA) or relaxed chromatin
migrates away from the nucleus. The extent of DNA liberated from the head of
the comet is directly proportional to the DNA damage.
Equipment
1. Electrophoresis unit
2. Power pack
3. Microscope slides
Preparation of Reagents
1. Lysing Solution (stock)
36.52gm of NaCl, 9.3 gm of EDTA, 0.3 gm of Tris are dissolved in 100 ml
of distilled water and stirred for 15min on the magnetic stirrer. 3gm of
sodium hydroxide is added to the mixture and allowed to dissolve. The pH
is adjusted to 10 with HCl or NaOH and the volume is made up to 250 ml.
The solution is stored at room temperature.
67
2. Lysing Solution (Working)
1% Triton-x and 10% DMSO is added to the stock lysing solution (89ml) to
make it up to 100ml and refrigerated for 30 minutes prior to use (pH 10).
3. Electrophoresis buffer
12 gms of NaOH, 0.36 gms of EDTA are dissolved in 1litre of distilled
water (pH –13).
3. Neutralizing buffer
48.5 gm of Tris is dissolved in 1000ml of distilled water to make 0.4M Tris
buffer (pH 7.5).
4. Fixing solution
75 gm of TCA, 25 gm of ZnSO4, 25gm of glycerol are added in 500 ml of
distilled water.
68
Methodology
2. Layering of gels
Pre coated slides are layered with a mixture of 110µ l of 0.5% LMPA and
20µ l of whole blood. The cover slip is placed and gel is allowed to set for
4 to 10 minutes on ice pack. The cover slip is slided off and then the slides
are layered with 110µ l of LMPA for the third layer. The cover slip is
placed on the gel and allowed to set for 4 –10 minutes on ice pack. The
cover slip is slided off.
4. Unwinding of DNA
The slides are taken from lysing solution and placed side by side in
horizontal electrophoresis unit. The electrophoretic chamber is filled with
alkaline buffer until the buffer level completely covers the slides. Bubble
formation is avoided. The slides are kept in buffer for 20 min to allow the
DNA for unwinding.
5. Electrophoresis
Turn on the power supply to 25 volts and current is adjusted to 300mA by
raising or lowering the buffer level. Electrophoresis is carried out for 20
min and the slides are lifted gently and placed in a tray.
69
The slides are flooded with 0.4M Tris neutralizing buffer for 10 min and the
buffer is drained off and the step is repeated twice.
7. Fixing the slides
The slides are placed in fixing solution for 10 minutes and washed for
several times with distilled water. The slides are air dried at room
temperature overnight.
10. Interpretation
DNA damage is estimated based on the tail length and percentage of
comets.
70
Good Laboratory Practices (GLP)
Safety Aspects
All laboratory staff should be well versed and responsible for proper
functioning of all the safety equipments and personal protective wares.
Do not keep more chemicals in the laboratory than necessary for
the ongoing work. Store the rest in a safe place.
Always wear laboratory coats and safety glasses. Eating, drinking
and smoking should be prohibited in the laboratory.
Ensure that safety devices and precaution manuals are easy to
find.
Take care of fire extinguishers, fume hoods, chemical spill kit, eye
washes and other safety devices. Always have a first-aid kit ready in
the laboratory.
Used liquids/chemicals should be disposed off in a proper way.
Follow the procedure precisely.
Check analytical procedures if they are not clear, especially while
working with organic liquids which may be dangerous.
Be careful with power supply, gas cylinders and heating
equipments.
71
Work as much as possible in a fume hood and always add
acid/base to water.
Don’t try to catch falling glassware. Remove and replace broken
glassware.
Laboratory staff should undergo an annual medical examination.
Safety procedures should be displayed as poster in the laboratory.
Some training for laboratory staff is necessary for handling
emergencies.
Laboratory Hazards
72
NaOH and certain other chemicals produce considerable heat on
dissolution, which may cause burns.
Some metals (arsenic, nickel, mercury) are highly toxic and may
also be carcinogenic. Avoid inhalation, ingestion and skin contact.
Nearly all organic solvents are hazardous. Some are probably
carcinogenic and should be treated with extra caution.
Avoid mouth pipetting. Use of auto pipettes is advisable.
Beware of physical hazards from electrical items and gas cylinders.
Always remember, HOT glassware looks exactly like COLD
glassware, be careful while handling.
73
Prepare data recording format ahead of time. Well prepared data
recording format not only speeds up the recording of data, but also
helps greatly during calculations and report writing.
Clean your glassware at the end of the lab period so that it is ready
for the next laboratory work.
Many people forget to write down their observations. Colour
changes, endothermic or exothermic changes, changes in physical
state, boiling point, melting point, freezing point, etc are very crucial to
record. Look at the data, do they look reasonable for the type of
experiment and expected results? When in doubt, repeat a portion of
the experiment, there is no better lesson than to find your own mistake.
If you are still unsure, ask the lab instructor.
Lab instructor will sometime discuss the important aspects of the
lab with students individually or in small groups in an effort to help
them get more out of the experiment.
Lastly, it is important to follow the safety do's and don'ts of the
laboratory, not only for your own safety but also for your fellow beings.
74