electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
5. Electrophoresis
DrAnurag yadav,Bio-FMMC5
a separation technique
Simple, rapid and highly sensitive
used in clinical laboratories to separate charged molecules from each
other in presence of electric field
– Proteins in body fluids: serum, urine, CSF
– Proteins in erythrocytes: hemoglobin
– Nucleic acids: DNA, RNA
6. Clinical applications of Electrophoresis
Serum Protein Electrophoresis
LipoproteinAnalysis
Diagnosis of Haemoglobinopathies and HaemoglobinA1c
Determination of Serum Protein Phenotypes and Micro
heterogeneities eg. α1- antitrypsin deficiency, MM
Genotyping of Proteins eg.ApoE analysis forAlzheimer’s disease
(polymorphic protein)
Small Molecules (Drugs, Steroids) Monitoring
Cerebrospinal FluidAnalysis
UrineAnalysis ( determination of GNs)
7. Principle :
DrAnurag yadav,Bio-FMMC7
Comprehensive term that refers to the migration of charged particle of
any size in liquid medium under the influence of an electric field.
Depending on kind of charge the molecule carry, they move towards
either
To cathode
Or toAnode
An ampholyte become positively charged in acidic condition and migrate
to cathode, in alkaline condition they become negatively charge and
migrate to anode.
8. DrAnurag yadav,Bio-FMMC8
Eg: as protein contain the ionizable amino and carboxyl
group.
The rate of migration of an ion in electrical field depend on
factors,
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5.Temperature of operation
9. 1. Mobility
DrAnurag yadav,Bio-FMMC9
Under the electrical field, the mobility of the particle is
determined by two factors:
Its charge
Frictional coefficient
Size and shape of the particle decide the velocity with which the
particle will migrate under the given electrical field and the
medium.
11. 2. Strength of electrical field
DrAnurag yadav,Bio-FMMC11
It determined by the force exerted on the particle, and the charge the particle
carrying.
F=QV
when force is exerted on the particle it start moving, however the moment is
restricted by the experience of the frictional force because of the viscosity.
12. Effect of pH on Mobility
DrAnurag yadav,Bio-FMMC12
As the molecule exist as amphoteric , they will carry the
charges based on the solvent pH.
Their overall net charge is NEUTRAL when it is at zwitter
ion state.And hence the mobility is retarded to zero.
Mobility is directly proportional to the magnitude of the
charge, which is functional of the pH of solvent.
The pH is maintained by the use of Buffers of different pH.
15. Power supply
DrAnurag yadav,Bio-FMMC15
Drives the moment of ionic species in the medium and allow
the adjustment and control of the current or voltage.
Constant delivery is required.
Pulsed power can also be applied.
16. Buffer
DrAnurag yadav,Bio-FMMC16
The buffer in electrophoresis has twofold purpose:
Carry applied electrical current
They set the pH as which electrophoresis is carried out.
Thus they determine;
Type of charge on solute.
Extent of ionization of solute
Electrode towards which the solute will migrate.
The buffer ionic strength will determine the thickness of the ionic
cloud.
17. Commonly buffers used;
DrAnurag yadav,Bio-FMMC17
Buffer pH value
Phosphate buffer around 7.0
Tris-Borate-EDTA buffer (TBE) around 8.0
Tris-Acetate EDTA buffer (TAE) above 8.0
Tris Glycine buffer (TG) more than 8.5
Tris -Citrate-EDTA buffer (TCE) around 7.0
Tris -EDTA buffer (TE) around 8.0
Tris -Maleic acid -EDTA buffer (TME) around 7.5
Lithium Borate - buffer (LB) around 8.6
18. Supporting medium
DrAnurag yadav,Bio-FMMC18
Supporting medium is an matrix in which the protein
separation takes place.
Various type has been used for the separation either on slab
or capillary form.
Separation is based on to the charge to mass ratio of protein
depending on the pore size of the medium, possibly the
molecular size.
19. Chemical nature inert
Availability easy
Electrical conductivity high
Adsorptivity low
Sieving effect desirable
Porosity controlled
Transparency high
Electro-endosmosis (EEO) low
Rigidity moderate to high
Preservation feasible
Toxicity low
Preparation easy
Properties:
21. Agarose Gel
DrAnurag yadav,Bio-FMMC21
A linear polysaccharide (made-up of repeat unit of agarobiose-alternating
unit of galactose and 3,6-anhydrogalactose).
Used in conc as 1% and 3%.
The gelling property are attributed to both inter- and intramolecular
hydrogen bonding
Pore size is controlled by the % of agarose used.
Large pore size are formed with lower conc and vice versa.
Purity of the agarose is based on the number of sulphate conc, lower the
conc of sulphate higher is the purity of agarose.
22. DrAnurag yadav,Bio-FMMC22
ADVANTAGES:
Easy to prepare and small
concentration of agar is required.
Resolution is superior to that of
filter paper.
Large quantities of proteins can be
separated and recovered.
Adsorption of negatively charged
protein molecule is negligible.
It adsorbs proteins relatively less
when compared to other medium.
Sharp zones are obtained due to less
adsorption.
Recovery of protein is good, good
method for preparative purpose.
DISADVANTAGES:
Electro osmosis is high.
Resolution is less compared to
polyacrylamide gels.
Different sources and batches of
agar tend to give different results
and purification is often necessary.
APPLICATION:
Widely used in Immuno
electrophoresis.
Gel Structure of Agarose:
23. Cellulose acetate
DrAnurag yadav,Bio-FMMC23
Thermoplastic resin made by treating cellulose with acetic
anhydride to acetylate the hydroxyl group.
When dry, membrane contain about 80% air space within fibers
and brittle film.
As the film is soak in buffer, the space are filled.
Because of their opacity, the film has to be made transparent by
soaking in 95:5 methanol:glacial acetic acid.
It can be stored for longer duration.
24. Polyacrylamide
DrAnurag yadav,Bio-FMMC24
Frequently referred to as PAGE.
Cross-linked polyacrylamide gel are formed from the polymerization of
the monomer in presence of small amount of N,N”-methylene-
bisacrylamide.
Bisacrylamide – two acrylamide linked by the methylene group.
The polymerization of the acrylamide is an example for free radical
catalysis.
They are defined in terms of total percentage of acrylamide present, and
pore size vary with conc.
25. DrAnurag yadav,Bio-FMMC25
Made in conc between 3-30% acrylamide.
Thus low % has large pore size and vice versa.
Proteins are separated on the basis of charge to mass ratio and
molecular size, a phenomenon called Molecular sieving.
ADVANTAGES:
Gels are stable over wide range of pH and temperature.
Gels of different pore size can be formed.
Simple and separation speed is good comparatively.
27. a. Electrophoresis Separation
DrAnurag yadav,Bio-FMMC27
When performed on precast or agarose gel, following steps
are followed;
- Excess buffer removed
- 5-7 μL sample
- Placed in electrode chamber
- Current application
- Gel is rinsed, fixed and dried
- Stained
- Scanned under densitometry
28. b. Staining
DrAnurag yadav,Bio-FMMC28
Protein is ppt in gel by using acetic acid or methanol
(this will prevent diffusion of protein out of the gel when
submerged in stain solution)
Amount of dye taken by sample is affected by many factors,
Type of
protein
Degree of
denaturation
29. Different stains of Electrophoresis
Plasma Proteins
- Amido black
- Coomassie Brilliant Blue
- Bromophenol Blue
Hemoglobins
- Amido black
- Coomassie Brilliant Blue
- Ponceau Red
Lipoproteins
- Sudan Black
DNA ( Fluorescent dyes)
- Ethidium Bromide
- Sybr Green, Sybr Gold
30. Staining Systems
Proteins
General – Coomassie brilliant blue R, Kenacid blue,Amido
black.
Specific – Oil red O, PAS, Rubeanic acid,Transferrin-specific & for
calcium binding proteins
Steps * fixing
* staining
* destaining
Allozymes - Histochemical staining
DNA - EtBr, SyBR green, Propidium iodide and
silver staining
31. C. Detection and Quantification
DrAnurag yadav,Bio-FMMC31
Once separated, protein may be detected by staining
followed by the quantification using the densitometer or by
direct measuring using an optical detection system under set
at 210nm.
Separation type Wavelength
Serum protein 520-640nm
Isoenzymes 570nm
Lipoproteins 540-600nm
DNA fragments 254-590nm
CSF protein ----
The selection of the wavelength is the property o type of stain used for the identification of
separation.
38. Common effect of variables on
separation
DrAnurag yadav,Bio-FMMC38
pH Changes charge of analyte, effective mobility; structure of analyte-
denaturing or dissociating a protein.
Ionic strength Changes in voltage; increased ionic strength reduces migration velocity
and increase heating.
Ions present Change migration speed; cause tailing of bands.
Current Too high current cause overheating.
Temperature Overheating cause denature protein; lower temp reduce diffusion but also
migration; there is no effect on resolution.
Time Separation of bands increases linearly with time, but dilution of bands
increase with square root of time.
Medium Major factors are endosmosis and pore size effect, which effect migration
velocities.
39. TYPES OF ELECTROPHORESIS
1) Zone Electrophoresis
a) Paper Electrophoresis
b) Gel Electrophoresis
c) Thin Layer Electrophoresis
d) Cellulose acetate Electrophoresis
2) Moving Boundary Electrophoresis
a) Capillary Electrophoresis
b) Isotachophoresis
c) Isoelectric Focussing
d) Immuno Electrophoresis
39
40. CLASSIFICATION
• Traditional methods, using a
rectangular gel regardless of
thickness
Slab gel
electrophoresis
• DISContinuities in electrophoretic
matrix caused by layers of
polyacrylamide/starch gel that
differ in composition & pore size
Disc
electrophoresis
41. CLASSIFICATION
• IEF separates amphoteric
compounds, such as proteins, with
increased resolution in a medium
possessing a stable pH gradient
Isoelectric
focusing
electrophoresis
• Completely separates smaller ionic
substances into adjacent zones tat
contact one another with no overlap
& all migrate at the same rate.
Isotachophoresis
42. CLASSIFICATION
• Power is alternately applied to different pair
of electrodes/ electrode arrays, so the
electrophoretic field is cycled b/w 2
directions.
Pulse-Field
electrophoresis
• Charge-dependent IEP in the first
dimension.
• Molecular weight dependent electrophoresis
in second.
2-D
electrophoresis
43. SUPPORT MEDIA IN SEPERATION
Molecular size
• Gradient gels
• Gels containing denaturants
Molecular size &
Charge
• Gel electrophoresis
• Immunoelectrophoresis
• 2D electrophoresis
47. Cellulose acetate electrophoresis
DrAnurag yadav,Bio-FMMC47
Although older, still has number of application.
Has advantage over paper, being homogenous medium with
uniform pore size and doesnot absorb the protein.
Much less tailing of the band.
Resolution is better than paper.
48. DrAnurag yadav,Bio-FMMC48
Much simpler to run. Can be used as single sample or
multiple sample run.
Acetate paper is first wetted in the buffer, and the sample is
loaded.
The strip is kept for the electrophoretic run.
6-8V/cm for about 3 hr.
The protein separation is stained, for better visualization.
49. DrAnurag yadav,Bio-FMMC49
Although used for the serum protein separation, but replaced
by the agarose gel ( which give better resolution).
The enzymes can easily detected by using Zymogram
technique.
53. SDS-PAGE
DrAnurag yadav,Bio-FMMC53
Sodium dodecyl sulphate- polyacrylamide gel
electrophoresis.
Most widely used method for analysing protein mixture
qualitatively.
Useful for monitoring protein purification – as separation of
protein is based on the size of the particle.
Can also be used for determining the relative molecular mass
of a protein.
54. DrAnurag yadav,Bio-FMMC54
Mercaptoethanol will break the disulphide bridges.
SDS binds strongly to and denatures the protein.
Each protein is fully denatured and open into rod-shape with
series of negatively charged SDS molecule on polypeptide chain.
SDS is an anionic
detergent.
The sample is first
boiled for 5min in
buffer containing
• Beta-
Mercaptoethanol
• SDS
55. DrAnurag yadav,Bio-FMMC55
On average, One SDS molecule bind for every two amino
acid residue.
Hence original native charge is completely swamped by the
negative charge of SDS molecule.
Also referred as Discontinuous gel electrophoresis.
57. DrAnurag yadav,Bio-FMMC57
Stacking gel: ordering/arranging and conc the
macromolecule before entering the field of separation.
(4% of acrylamide)
• Purpose is to concentrate protein sample in sharp band before enters
main separating gel.
Running gel: the actual zone of separation of the
particle/molecules based on their mobility. (15% of
acrylamide)
Pore size: routinely used as 3% to 30% which is of pore
size 0.2nm to 0.5nm resp.
61. DrAnurag yadav,Bio-FMMC61
In separating gel, protein separate owing to molecular sieving
properties.
Smaller proteins pass more easily, larger one retarded by
friction.
62. - Research tool
- Measuring molecular weight
- Peptide mapping
- Protein identification
- Determination of sample purity
- Identifying disulfide bonds
- Separation of proteins and establishing size
- Blotting
- Smaller fragments of DNA
- Separation of nucleic acids
- Major clinical use –ALP separation
APPLICATION:
63. ADVANTAGES:
- Clear, fairly easy to prepare
- Exhibit reasonable mechanical strength over acrylamide conc
- Low endosmosis effect
DISADVANTAGES
- Gel preparation and casting- exacting n time-consuming
- Complete reproducibility of gel preparation not possible
64. STAINING:
Fluorescent stains - Ethidium bromide – Nucleic acids
Silver stain for protein gel (sensitive 50 times dye based)
Dye based – Coomassie blue – 50ng protein band
Tracking dyes – BPB> xylene cyanol, Orange G
65. Native (buffer) gel
DrAnurag yadav,Bio-FMMC65
Done by using the polyacrylamide gel (7.5%).
As used for the enzyme separation, the denaturing agent is
not added - hence SDS is absent.
pH of 8.7
Proteins are separated according to the electrophoretic
mobility & Sieving effect of the gel.
66. DrAnurag yadav,Bio-FMMC66
Alternative approach for enzyme detection is to include the
substrate on agarose gel, which is poured over acrylamide gel.
The diffusion and interaction of the substrate and the enzyme
results in color formation.
This can be cut and used for
Total protein estimation
Enzyme activity.
67. Gradient gel
DrAnurag yadav,Bio-FMMC67
This is again an polyacrylamide gel system.
Instead of running a slab of uniform pore size, a gradient gel
is formed.
Uniformly from 5% to 25% acrylamide from top to bottom.
The highest conc gradient is layed first and than decreasing
gradient is poured.
But the sample move down, were the pore size reduces along
the path.
68. DrAnurag yadav,Bio-FMMC68
Normally run with the stacking gel at the top.
Advantage :
Greater range of protein can be separated. (Complex mixtures
can be run.)
Protein with similar molecular range may be resolved.
Protein moves till the pore size become smaller n limit its
descend further.
Proteins separated will have a distinct sharp bands.
69. Isoelectric focussing gels
DrAnurag yadav,Bio-FMMC69
First described by- H.Svensson in Sweden.
Method is ideal for the separation of the amphoteric
substances.
Method has high resolution.
Able to separate the protein which differ in isoelectric point
by little 0.01 of pH unit.
Most widely used as the horizontal gel slab.
71. Establishment of ph gradient:
DrAnurag yadav,Bio-FMMC71
This is achieved by the ampholyte & must have following prop:
Must dictate pH course (buffering capacity at their Ip)
Should have conductance at their Ip.
Low molecular weight
Soluble in water
Low light absorbance at 280nm.
Available commercially with pH band (3-11)
Eg:Ampholine, Pharmalyte and Bio-lyte.
74. DrAnurag yadav,Bio-FMMC74
Duration : 2-3h
High voltage : 2500V
Cooling plates : 100C
Stable power pack
Fixing (trichloroacetic acid) and Staining (Coomassie
Brilliant blue)
75. DrAnurag yadav,Bio-FMMC75
Application:
- Highly sensitive for studying the microheterogeneity of
proteins
- Useful for separating the isoenzymes.
- Human genetic lab
- Research in enzymology, immunology,
- Forensic, food and agriculture industry,
76. Two-dimensional polyacrylamide
gel electrophoresis
DrAnurag yadav,Bio-FMMC76
Principle :
Technique combines with
IEF as first dimensional.
• Which separate according to
the charge.
Second dimension by
SDS-PAGE
• Separate according molecular
size.
77. DrAnurag yadav,Bio-FMMC77
Thus combination gives sophisticated
analytical method for analysing the
protein mixture.
Size very from 20*20cm to the minigel.
IFE is carried on acrylamide gel
(18cm*3mm), with 8M urea.
After separation, placed on 10% SDS-
PAGE for further separation .
78. DrAnurag yadav,Bio-FMMC78
Used in field of proteomics.
Can separate 1000 to 3000 proteins from the cell or an tissue
extract.
80. Isotachophoresis
DrAnurag yadav,Bio-FMMC80
Used for separation of smaller ionic substances.
They migrate adjacent with contact one another, but not
overlapping.
The sample is not mixed with the buffer prior to run.
Hence current flow is carried entirely by the sample ions.
Faster moving ions migrate first and the adjacent ones next
with no gap between the zone .
81. DrAnurag yadav,Bio-FMMC81
All ions migrate at the rate of fastest ion in zones.
Then it is measured by UV absorbance.
Application-
Separation of small anions and cations
Amino acids
Peptides
Nucleotides
Nucleosides
Proteins.
82. Pulsed-Field Electrophoresis
DrAnurag yadav,Bio-FMMC82
Power is applied alternatively to different pair
of electrodes
Electrophoretic field is cycled at 105-1800
Because of which the molecule have to orient
to the new field direction
This permit separation of large molecule like
DNA .
Applied: for typing various strain DNA.
83. High voltage electrophoresis
DrAnurag yadav,Bio-FMMC83
First described by Michl.
As the name describe, the electrophoresis is carried under
the very high voltage.
This is required for the substances of lower molecular weight
which will have considerable high diffusion rate.
Eg: amino acids, peptides.
84. DrAnurag yadav,Bio-FMMC84
The voltage applied was ranging from
2500-10000V or
50-200V/cm, 500mA.
This resulted in better resolution and even very rapid
separation.
And even with tremendous amount of heat generation.
To tackle this, it need a good cooling system.
86. DrAnurag yadav,Bio-FMMC86
Precautions :Temperature of the system has to be maintained
constant.
Plate dimension 50*50cm
The HVE one direction can be combined with the
chromatography- which is right angle to first.
Possible even to run in two direction at two different pH.
87. Capillary electrophoresis
DrAnurag yadav,Bio-FMMC87
Technique first described by- Jorgensen and Lukacs (1980’s)
Also referred as
High performance capillary electrophoresis(HPCE)
Capillary zone electrophoresis (CZE)
Free solution capillary electrophoresis (FSCE)
Capillary electrophoresis (CE)
88. DrAnurag yadav,Bio-FMMC88
The sensitivity has made it as one of the choice for many
biomedical and clinical analyses.
Application : used to separate
Amino
acids
Peptides Proteins
DNA
fragments
Nucleic
acid
Drugs /
even
metals.
92. High voltage injection Pressure injection
DrAnurag yadav,Bio-FMMC92
The buffer reservoir is
replaced by the sample
reservoir the high
voltage is applied (+
electrode) buffer
reservoir is placed again
and voltage applied for the
separation.
Anodic end of capillary is
removed from buffer and
placed in air tight sample
sol with pressure sample
is pushed into capillary
kept back in the
buffer sample and voltage
is applied.
Sample application is done by either of one method
High voltage
injection
Pressure
injection
93. DrAnurag yadav,Bio-FMMC93
50μm – ID.
300 μm – ED.
Length – 50-100cm.
Fused silica capillary tube.
Polyimide coating external.
Packed with the buffer in use.
As the name suggest, the separation is carried in a narrow
bore Capillary
94. DrAnurag yadav,Bio-FMMC94
High voltage is applied (up to 50 kV)
The components migrate at different rate along the length.
Although separated by the electrophoretic migration, all the
sample is drawn towards cathode by electroendosmosis.
Since this flow is strong, the rate of electroendosmotic
flow is greater than the electrophoretic velocity of the
analyte ion, regardless of the charge.
96. DrAnurag yadav,Bio-FMMC96
DETECTION:
near to cathode end,
viewing window
- Detected by the
ultraviolet monitor,
transmit signal and
integrated by
computer.
- Refractive index
- Fluorescence
- CE-MS
97. DrAnurag yadav,Bio-FMMC97
Troubleshooting :
Adsorption of protein to the wall of capillary – leading to
smearing of protein – viewed as peak broadening – or complete
loss of protein.
- Use of neutral coating group to the inner surface of the capillary.
98. DrAnurag yadav,Bio-FMMC98
Advantage over slab type:
Reduce the problem of heating effect.
Large surface to volume ratio.
Less diffusion of the separated bands.
99. DrAnurag yadav,Bio-FMMC99
Variations in technique:
Add of surfactant to buffer i.e., SDS (for Neutral molecules).
Micellar formation In MECC- electrophoresis + chromatography.
100. Different modes of operation
DrAnurag yadav,Bio-FMMC100
Capillary zone electrophoresis :
- Separation principle based on charge to mass ratio of
molecule.
- Separation is faster.
- Due to High EOF, the molecules regardless of the charge,
they are moved to cathode.
101. Different modes of operation
DrAnurag yadav,Bio-FMMC101
Micellar electrokinetic chromatography:
- It is an hybrid.
- Used for separation of the neutral and charged solutes.
- The separation is accomplished by micelles formation. (8-
9mmol/L for SDS)
- During migration, micelle interact with analyte as
chromatographic manner and the separation is brought
about.
103. Different modes of operation
DrAnurag yadav,Bio-FMMC103
Capillary gel electrophoresis:
- Identical to the slab.
- Separation based on the sieving.
- The capillary is filled with “sieving matrix” or “soluble
polymer network”.
- Low viscosity, self entangling for formation of pore size.
- Variety of polymeric matrices are available for DNA and Protein.
- Cross linked polyacrylamide- choice of polymer.
104. DrAnurag yadav,Bio-FMMC104
Advantage over
conventional
• Online detection.
• Improved quantification.
• Almost complete automation.
• Reduced analysis time.
• Wider choice of gel matrices.
• Linear polyacrylamide, derivative of
cellulose, galactomannan,
glucomannan, polyvinyl alcohol,
polyethyleneoxide, agarose, dextran,
polymethylacrylamide, and
polyacryloylethoxyethenol.
105. Different modes of operation
DrAnurag yadav,Bio-FMMC105
Capillary isoelectric Focussing Electrophoresis:
- Is comparable to tube IEF.
- Carried out in the capillary.
- The focused zone migrate to the detector with the separated
sample.
- cIEF is completed in ~15 min.
106. Different modes of operation
DrAnurag yadav,Bio-FMMC106
Capillary Isotachophoresis:
- Same feature as ITP.
- Except condition of pure ITP not achieved.
- Typically used for online sample preconcentration.
- CZE, MEKC, CGE.
107. DrAnurag yadav,Bio-FMMC107
a. Capillary Isotachophoresis
b. Capillary gel electrophoresis
c. Capillary isoelectric Focussing
Electrophoresis
d. Micellar electrokinetic
chromatography
summary
108. Capillary Electrophoresis (CE) versus High
Performance Liquid Chromatography (HPLC)
DrAnurag yadav,Bio-FMMC108
CE has flat flow, compared to pumped parabolic flow of HPLC.
Flat flow will have narrower peaks & better resolution.
CE has greater peak capacity.
109. DrAnurag yadav,Bio-FMMC109
HPLC is more thoroughly developed.
HPLC is more complex than CE.
HPLC has wider variety of column length and packing
Both techniques uses similar modes of detection.
Can be used complementary to one another.
111. Microchip electrophoresis
DrAnurag yadav,Bio-FMMC111
Current advanced method.
Development in technique include
Integrated microchip design
Advanced detection system
New application
Protein and DNA separation can be done
112. Instrumentation
DrAnurag yadav,Bio-FMMC112
Similar to the capillary electrophoresis.
Separation channel
Sample injection (50-100pL)
Reservoirs
Voltage (1-4kV)
sample preparation
Precolumn or postcolumn reactors.
Classical Cross-T design.
Time period of 50-200sec.
114. Application
DrAnurag yadav,Bio-FMMC114
An alternative for the DNA analysis.
Herpes simplex virus DNA in CSF for diagnosing encephalitis.
Gene rearrangement correlative with lymphoproliferative
disorders.
Polymorphisms in gene.
Tetranucleotide associated with hypercholesterolemia.
Diagnosing fragile X syndrome.
Muscular dystrophy.
Anthracis specific PCR product.
116. References
DrAnurag yadav,Bio-FMMC116
KeithWilson- Principles and techniques of biochemistry
and molecular biology.
Upadhyay- biophysical chemistry.
Tietz-Text book of clinical chemistry.
Kaplan- clinical chemistry.
YouTube and Google images.