DNA and Protein Analysis Methods Presentation

A presentation on
PCR, DNA Sequencing, DNA chips, DNA Microarray,
UV, IR, NMR, Mass Spectroscopy, their methods, and
Applications.
Presented by,
Md. Ashaduzzaman Nur
Dept. of Genetic Engineering & Biotechnology
Jashore University of Science & Technology

Outline:
Definition, Working procedure, & applications of
 PCR,
DNA sequencing,
DNA Microarray,
• UV Spectroscopy,
• IR Spectroscopy,
• NMR Spectroscopy,
• Mass Spectroscopy.

What is PCR?
• PCR is an in vitro technique for the amplification of a region of DNA
which lies between two regions of known sequence.
• PCR can make billions of copies of a target sequence of DNA in a
few hours.
• It was invented in 1983 by Dr. Kary Mullis, for which he received
the Nobel Prize in Chemistry in 1993.

Components needed for a PCR
1. Template (DNA/RNA/Plasmid)
2. Forward and Reverse Primer
3. dNTPs (Mixture of dATP, dTTP, dCTP, dGTP)
4. Polymerase
5. Polymerase buffer
6. MgCl2
7. Water

Types of PCR
Real Time PCR:
RT-PCR also known as quantitative PCR is used to amplify and
simultaneously quantify a target DNA. It differs from standard PCR in a
way that it can detect the amplified product as the reaction progresses
with time but in standard PCR the amplified product is detected at the
end of the reaction by agarose gel electrophoresis.
Use: Determining gene or transcript numbers present within different
samples

Types of PCR Cont….
Reverse Transcriptase PCR (RT-PCR):
A laboratory technique for amplifying a defined piece of a RNA
molecule.
Is amplify RNA sequences (especially mRNA) through synthesis of
cDNA by reverse transcriptase (RT). Subsequently, this cDNA is
amplified using PCR.
Use: Diagnosis of RNA viruses, study of gene expression in vitro.

Nested PCR:
Double process of amplification that increases the sensitivity due to
small amounts of the target are detected by using two sets of primers.
• The first primer pair amplified a region as in any PCR
• The second primer pair is located within the first PCR product and
amplified a PCR product that will be shorter than the first one
Use: Specificity of the first PCR product is verified with the second one.

Inverse PCR Steps:
• Target DNA is lightly cut into smaller fragments of several kilobases by
restriction endonuclease digestion.
• Self-ligation is induced under low concentrations causing the phosphate
backbone to reform. This gives a circular DNA ligation product.
• Target DNA is then restriction digested with a known endonuclease. This
generates a cut within the known internal sequence generating a linear
product with known terminal sequences. This can now be used for PCR
(polymerase chain reaction).
• Standard PCR is conducted with primers complementary to the now known
internal sequences

Multiplex PCR:
Is an adaptation of PCR which allows simultaneous amplification of
many sequences.
Use: Detect different pathogens & diagnosis of diseases in the same,
single sample.

Colony PCR:
Colony PCR is used for the screening recombinants from bacterial,
bacteriophage or yeast transformation products.

Steps of colony PCR:
Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette
tip from a growth plate.
Swirl it into 25micro l of TE buffer with autoclaved in an micro centrifuge tube
Heat the mix in boiling water bath at 90-100°C for 2 minutes
Again centrifuge it at 6000 rpm Collect the supernatant. Take 1-2 micro l of it and it
is used as template in a 25micro l PCR tube
Conduct standard PCR

Significance of Colony PCR:
• Colony PCR is a fast and reliable method for the screening of
recombinants.
• A no of colonies or plaques assayed simultaneously and there is no
need to store large no of transformed clones for long periods.
• This method used for cDNA library screening.

Touchdown PCR:
• This type of PCR is used to optimize yield of amplified product at different
annealing temperature.
• It is very difficult to find out the annealing temperature when there is
mismatches between primers and the template strands.
• Amplification of the specific target DNA starts when optimum Ta is achieved.
• The higher temperatures - greater specificity for primer binding
• lower temperature - more efficient amplification from the specific products
formed during the initial cycles
• It is the quickest method to optimize PCR when it is required to use new
template and primer combinations.
• Nowadays, modern PCR machine which have the facility of gradient setting are
easily programmed to run Touchdown PCR.

Asymmetric PCR
• In standard PCR, amplification of ds DNA occurs exponentially during the
early stages of PCR, but in the end slows down and plateau is formed because
of –ve feed back between the ds products and the Taq DNA polymerase.
• The plateau value in standard PCR is unsuitable for end point analysis of
starting target numbers.
• In asymmetric PCR, the end product is a single stranded DNA as a result of
unequal primer concentration.
• PCR is carried out as usual, but with a great excess of the primer for the strand
targeted for amplification.
• As asymmetric PCR proceeds, the lower concentration primer is quantitatively
incorporated into the ds DNA.
• The higher concentration of primer continues synthesis of DNA out of the
template strand in a linear amplification.

Use of Asymmetric PCR
• Asymmetric PCR is useful in end point analysis.
• The advanced form of asymmetric PCR, Linear-After The-Exponential
PCR(LATE PCR) uses a limiting primer and a excess primer that
differ 10-50 folds in their relative concentration.

Preparing a PCR Reaction Mixture (10µl)

How to write a PCR Program in a Thermal Cycler
• Denaturation
• Primer annealing
• Extension
• Final extension
• Hold
Step 1. 94-96º C 1-2 min/10-15 min
Step 2. 94-96º C 30 sec to 1 min
Step 3. 50-70º C 30 Sec to 1 min
Step 4. 72º C (68º C) 1 kb/min
Step 5. Go to step 2 34 more times
Step 6. 72º C 10 min
Step 7. Hold at 16ºC/4°C

Things to consider for Designing primers
• Length : variable (I prefer 18-30 bp)
• Primer Melting Temperature : Between 55 – 65º C
• GC Content : Above 40 % not more than 60%
• GC Clamp : 2-3 G or C at the 3’ end of the primer
• Primer Secondary Structures : No Hairpin loops,
Primer dimer, cross dimer

1. Primer length
2. Primer Melting Temperature: Primer Melting Temperature (Tm) by
definition is the temperature at which one half of the DNA duplex will
dissociate to become single stranded and indicates the duplex stability.
Primers with melting temperatures in the range of 52-58°C generally produce
the best results. Primers with melting temperatures above 65°C have a
tendency for secondary annealing. The GC content of the sequence gives a
fair indication of the primer Tm. The basic formulae for calculating Tm is:
Tm = 81.5 +16.6(log10[Na+]) + 0.41(%G+C) – 675/n
Where [Na+] is the molar salt concentration
n = number of base in the oligonucleotide
The simplest Tm formulae:
Tm = 4°C x (number of G’s and C’s in the primer) +
2°C x (number of A’s and T’s in the primer)
5’ – tcg atc gta atc gta cgt agt gca ctg g - 3’

3. Primer annealing temperature : The primer melting temperature is the
estimate of the DNA-DNA hybrid stability and critical in determining the
annealing temperature. Too high Tm will produce insufficient primer-template
hybridization resulting in low PCR product yield. Too low Tm may possibly lead
to non-specific products caused by a high number of base pair mismatches,.
Mismatch tolerance is found to have the strongest influence on PCR specificity.
4. GC Content : The GC content (the number of G's and C's in the primer as a
percentage of the total bases) of primer should be 40-60%.
5. GC Clamp : The presence of G or C bases within the last five bases from the
3' end of primers (GC clamp) helps promote specific binding at the 3' end due to
the stronger bonding of G and C bases. More than 3 G's or C's should be avoided
in the last 5 bases at the 3' end of the primer.

6. Primer Secondary Structures : Presence of the primer secondary structures
produced by intermolecular or intramolecular interactions can lead to poor or no yield
of the product. They adversely affect primer template annealing and thus the
amplification. They greatly reduce the availability of primers to the reaction.
I. Hairpins : It is formed by intramolecular interaction within the primer and should
be avoided.
II. Self Dimer : A primer self-dimer is formed by intermolecular interactions between
the two (same sense) primers, where the primer is homologous to itself. Generally a
large amount of primers are used in PCR compared to the amount of target gene.
When primers form intermolecular dimers much more readily than hybridizing to
target DNA, they reduce the product yield.
III. Cross Dimer : Primer cross dimers are formed by intermolecular interaction
between sense and antisense primers, where they are homologous. Optimally a 3'
end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of
-6 kcal/mol is tolerated generally.

7. Repeats : A repeat is a di-nucleotide occurring many times consecutively and
should be avoided because they can misprime. For example: ATATATAT. A
maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-
nucleotides.
8. Runs : Primers with long runs of a single base should generally be avoided as
they can misprime. For example, AGCGGGGGATGGGG has runs of base 'G' of
value 5 and 4. A maximum number of runs accepted is 4bp.
9. 3' End Stability : It is the maximum ΔG value of the five bases from the 3'
end. An unstable 3' end (less negative ΔG) will result in less false priming.

PCR procedure:
• The PCR consists of three steps which were repeated for 30-40 cycles.

Cycling Reactions:
1. Denaturation at 94°C :
During the heating step (denaturation), the reaction mixture is heated to
94°C for 1 min, which causes separation of DNA double stranded. Now,
each strand acts as template for synthesis of complimentary strand.
2. Annealing at 54°C :
This step consist of cooling of reaction mixture after denaturation step to
54°C, which causes hybridization (annealing) of primers to separated
strand of DNA (template).

3. Extension at 72°C : The reaction mixture is heated to 72°C which is
the ideal working temperature for the Taq polymerase. The polymerase
adds nucleotide (dNTP's) complimentary to template on 3’ –OH of
primers thereby extending the new strand.
4. Final hold: First three steps are repeated 35-40 times to produce
millions of exact copies of the target DNA. Once several cycles are
completed, during the hold step, 4–15 °C temperature is maintained for
short-term storage of the amplified DNA sample.

Emulsion PCR:
• Emulsion PCR is a PCR variation that some NGS technologies use to
replicate DNA sequences. It is conducted on a bead surface within tiny
water bubbles floating on an oil solution.
Principle:
• The basic principle of emulsion PCR is dilution and
compartmentalization of template molecules in water droplets in a
water-in-oil emulsion. Each droplet contains a single template
molecule and functions as a micro-PCR reactor.

Procedure of Emulsion PCR:
1. Fragmentation of DNA:
• The sample is fragmented ranging from 300 to 800bp.
2. Ligation of adapters:
• Adapters with one end sticky and one blunt are ligated with fragments.
• Phosphates are removed from sticky ends to avoid dimerization.

3. Formation of clonal bead populations:
• Beads coated with streptavidin are used.
• Beads have primers that matches the adapters used.
• Each bead is emulsified in a water-in-oil droplet with PCR reagents
(DNA polymerase, primers, buffers, dNTPs).

4. Amplification:
Denaturation to single strands:
• The double stranded DNA's with adapters are then denatured by heating the
DNA up to 95 °C.
Annealing of ssDNA:
• The ssDNA is then attached to the beads.
• Reverse strand (bottom strand or 3’-5’ strand) anneal to the f-primer on
bead surface and r-primer anneal to the forward strand (top strand or 5’-3’
strand).
Extension:
• Polymerase amplifies the forward strand (5’-3’) starting from beads towards
the primer site.
• The reverse strand is amplified starting from primer towards the bead site.

Cycle:
• Each newly formed double-strand is denatured, allowing for the strand to
ligate to another site on the surface of the bead. Eventually, 1 million copies
of the target is amplified on the surface of each bead.
5. Emulsion Breaking:
• After amplification, the emulsions are broken using isopropanol and
detergent buffer. The solution is then vortexed, centrifuged, and
magnetically separated.

6. Bead enrichment:
• After PCR is conducted, you are left with a mixture of some beads that have
amplified DNA attached on its surface, and some that do not.
• We may take out the enriched beads by attaching streptavidin coated
magnetic enrichment bead. With a magnet, we can then pull out the beads
with amplified DNA.
7. Bead Capping:
• Attach a capping oligonucleotide to the 3' end of both unextended forward
ePCR primers. This helps in coverslip arraying, which is used to polony
sequencing, and prevents fluorescent probes from ligating to the ends.
8. Result:
• The beads with amplified sequences are then placed on a slide and are
sequenced. Due to their high density of the same DNA molecule, the signal
is amplified, allowing computers to read the sequencing data.

POLYMERASE CYCLING ASSEMBLY (PCA) PCR:
• This entails the artificial synthesis of long DNA sequences by performing
PCR on a pool of long oligonucleotides with short
overlapping segments.
• The oligonucleotides alternate between sense and antisense directions,
and the overlapping segments determine the order of the PCR fragments,
thereby selectively producing the final long DNA product.

How Does Polymerase Cycling Assembly Work?
• PCA is pretty much like PCR, which is super handy! The main PCA
protocol is a Two-Step Assembly, and whether or not you need the
second step comes down to the size of your final product.
• The goal of Step 1 in the Two-Step Assembly is to piece together
sequences up to approximately 1kb. To begin, you must first:
(1) figure out the order of genes you want to stitch together.
(2) design primers (forward and reverse) that will result in a 20bp
overlapping sequence with the adjacent piece of DNA.
(3) generate these DNA fragments by PCR (common end size is ~
500bp).

Advantages of PCR:
Extremely high sensitivity
Specific amplification
Easy to set up
Rapid
Post-PCR processing of products
Disadvantages of PCR:
Extremely liable to contamination
High equipment cost
High test cost
Setting up and running requires high technical skills
A positive result may be difficult to interpret

Applications of PCR
1.Molecular biological research
2.Genetic mapping studies
3.Clinical and diagnosis uses
4.DNA fingerprinting
5.Bioinformatics

Definition of DNA Sequencing
• DNA sequencing is the process of determining the sequence of
nucleotide bases in a piece of DNA.
• DNA sequencing helps in finding the order of nucleotide in DNA.

What are the purpose of DNA sequencing?
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms
• Functional and comparative genomics

DNA sequencing methods
• Historically there are two main methods of DNA sequencing:
1. Maxam and Gilbert method
2. Sanger method
Modern sequencing equipment uses the principles of the Sanger
technique.

Maxam & Gilbert methods
• Maxam–Gilbert sequencing is a method of DNA sequencing
developed by Allan Maxam and Walter Gilbert in 1976-1977.
• The sequence of a double-stranded or single-stranded DNA molecule
is determined by treatment with chemicals that cut the molecule at
specific nucleotide positions.

Principle of Maxam & Gilbert methods
Reaction in two stages:
• Chemical modification of the bases
• Modified base is removed from its sugar, pyperidin cleaves
phosphodiester bonds 5’ and 3’ and base is released

Procedure of Maxam & gilbert methods
• Denature a double-stranded DNA to single-stranded by increasing
temperature.
• Radioactively label one 5’ end of the DNA fragment to be sequence by
a kinase reaction using gamma -32p.
• Cleave DNA strand at specific positions using chemical reactions.
• For example; we can use one of two chemicals followed by pyperidin.
Dimethyl sulphate selectively attacks purine (A and G) while
hydrazine selectively attacks pyrimidines (C and T). The chemical
treatments outlined in maxam- Gilbert’s paper cleaved at G, A+G, C,
and C+T.
• Now in four reaction tubes, we will have several differently sized
DNA strands.

Advantages of Maxam & Gilbert methods:
• No premature termination due to DNA sequencing.
• Stretches of DNA can be sequenced which can not be done with
enzymatic method.
• Purified DNA can be read directly.
• Can be used to analyze DNA-protein interactions.
• Can be used to analyze nucleic acid structures.
Disadvantages of Maxam & Gilbert methods:
• Not widely used.
• Use of radioactively and toxic chemicals.
• It requires extensive use of hazardous chemicals.
• It has a relatively complex set up.
• It is difficult to scale up.

What is Sanger method:
• Sanger method also known as the chain termination method, is a
technique for DNA sequencing based upon the selective incorporation
of chain-terminating dideoxynucleotides by DNA polymerase during
in vitro DNA replication.
• It was developed by Frederick Sanger and colleagues in 1977.

Principle of Sanger method
• A DNA primer is attached by hybridization to the template strand and
deoxynucleotides triphosphates (dNTPs) are sequentially added to the
primer strand by DNA polymerase.
• The primer is designed for the known sequence at 3’ end of the
template strand.
• M13 sequence is generally attached to 3’end and the primer of this
M13 is made.
• The reaction mixture also contains dideoxynucleoside triphosphates
along with usual dNTPs.
• If during replication ddNTPs is incorporated instead of usual dNTPs in
the growing DNA strand then the replication stops at that nucleotide.
• Respective ddNTPs of dNTPs terminates chain at their respective site.
For example, ddATP terminates at A site, similarly ddCTP, ddGTP, and
dd TTP terminates at C, G, and T site respectively.

Requirements of Sanger method
DNA sequencing is performed in four separate tubes,
each containing
i. Single stranded DNA to be sequenced
ii. DNA polymerase
iii. Primers
iv. The four dNTPs (dATP, dCTP, dTTP and dGTP)
v. Small amount of one of the four ddNTPs
(ddATP or ddCTP or ddTTP or ddGTP)

Procedure of Sanger method
The First Step:
• First, many copies of the DNA
fragment are needed to be made.
• To do this, scientist use the Polymerase
Chain Reaction that heats and cools
DNA to make quick copies of the
fragment

Procedure of Sanger method cont…
The Next Step:
• Fragments are heated again to be
unwound in to single-stranded DNA
• Then a primer is added which binds
to the DNA
• ddNTPs are added
The Final Step:
The DNA strand is run through Gel
Electrophoresis which sorts the
fragments by size.

Comparison between Sanger method and Maxam-
Gilbert method

Next Generation Sequencing:
• Next-generation sequencing (NGS), also known as high throughput
sequencing, is the catch-all term used to describe a number of different
modern sequencing technologies including:
Illumina (Solexa) sequencing
Roche 454 sequencing
SOLiD sequencing
 Pyrosequencing

Overview of Next Generation Sequencing Protocol:
1.Library preparation
2.Clonal amplification
3.Cyclic array sequencing
DNA fragmentation
and invitro adaptor ligatio
sequencing
Bridge PCREmulsion PCR
Pyrosequencing Sequencing-by-ligation sequencing-by-synthesis
1
2
3
454 sequencing SOLID platform Solexa technology

Iilumina / SolexaSequencing
• In NGS, vast numbers of short reads are sequenced in a single
stroke.
• To do this, firstly the input sample must be cleaved into short
sections. The length of these sections will depend on the particular
sequencing machinery used.
• In Illumina sequencing, 100-150bp reads are used. Somewhat
longer fragments are ligated to generic adaptors and annealed to a
slide using the adaptors. PCR is carried out to amplify each read,
creating a spot with many copies of the same read. They are then
separated into single strands to be sequenced.
• The slide is flooded with nucleotides and DNA polymerase. These
nucleotides are fluorescently labelled, with the colour corresponding
to the base. They also have a terminator, so that only one base is
added at a time.

Iilumina / Solexa Sequencing
• An image is taken of the slide. In
each read location, there will be a
fluorescent signal indicating the
base that has been added.
• The slide is then prepared for the
next cycle. The terminators are
removed, allowing the next base
to be added, and the fluorescent
signal is removed, preventing the
signal from contaminating the
next image.

• The process is repeated, adding one nucleotide at a time and
imaging in between computers are then used to detect the base at
each site in each image and these are used to construct a sequence.

• All of the sequence reads will be the same length, as
the read length depends on the number of cycles
carried out.

Definition of Pyrosequencing:
 A method of DNA sequencing based on the “sequencing
by synthesis" principle.
 It differs from Sanger sequencing, relying on the detection
of pyrophosphate release (hence the name) on nucleotide
incorporation, rather than chain termination with
dideoxynucleotides.
 ssDNA template is hybridized to a sequencing primer.
 Incubated with the enzymes DNA polymerase, ATP
sulfurylase, luciferase and apyrase, and with the
substrates adenosine 5´ phosphosulfate (APS) and
luciferin.

Pyrosequencing:
 The addition of one of the four deoxynucleotide triphosphates(dNTPs)(in
the case of dATP we add dATPαS which is not a substrate for a luciferase)
initiates the second step.
 DNA polymerase incorporates the correct, complementary dNTPs onto
the template.
 This incorporation releases pyrophosphate (PPi)
stoichiometrically.
 ATPsulfurylase quantitatively converts PPi to ATP in the presence of
adenosine 5´ phosphosulfate.
 This ATPacts as fuel to the luciferase-mediated conversion of luciferin
to oxyluciferin that generates visible light in amounts that are
proportional to the amount of ATP.
 The light produced in the luciferase-catalyzed reaction is detected by a
camera and analyzed in a program.
 Unincorporated nucleotides and ATPare degraded by the apyrase, and
the reaction can restart with another nucleotide.

Advantages of NGS:
1. Construction of a sequencing library for clonal amplification to
generate sequencing features.
2. No in-vivo cloning, transformation, colony picking.
3. Array-based sequencing.
4. Higher degree of parallelism than capillary-based sequencing.

Capillary Electrophoresis:
• These kind of separations are facilitated by the use of high voltages, which
may generate electro-osmotic and electro-phoretic flow of buffer solutions
and ionic species, respectively within the capillary.
Principle:
• Capillary electrophoresis is an analytical technique that separates ions
based on their electrophoretic mobility with the use of an applied voltage.
The electrophoretic mobility is dependent upon the charge of the molecule,
the viscosity, and the atom's radius. The rate at which the particle moves is
directly proportional to the applied electric field i.e. the greater the field
strength, the faster the mobility & vice versa.

• Neutral species are not affected, only ions move with the electric field.
If two ions are the same size, the one with greater charge will move
the fastest. For ions of the same charge, the smaller particle has less
friction and overall faster migration rate. Capillary electrophoresis is
used most predominately because it gives faster results and provides
high resolution separation. It is a useful technique because there is a
large range of detection methods available.

Instrumentation:
A typical capillary electrophoresis system consists of a:
•Buffer solution (like sodium dihydrogen phosphate,NaH2 PO4).
•High-voltage power supply (5 to 30 kv).
•A sample introduction system / sample injection (by pressure or vacuum).
•A capillary tube with internal diameter of 10-100mm & 20-100cm length.
•A detector.
•Output device.
Some instruments include a temperature control device to ensure reproducible
results. This is because the separation of the sample depends on the electrophoretic mobility
and the viscosity of the solutions decreases as the column temperature rises.

Sample Injection:
• Hydrodynamic injection: By applying pressure, by applying vacuum
and by gravitation
• Electrokinetic injection: By using Electric supply.
Detectors:
•Detectors similar to those used in GC,HPLC.
•Majority of instruments have UV detectors available.
• Alternative detector modes include commercially available IR,
fluorescence, laser induced fluorescence, conductivity and indirect
detection.
• The mass spectrometers is frequently used to give structural
information on the resolved peaks.

Capillary gel electrophoresis (CGE):
i.CGE is the adaptation of traditional gel electrophoresis into the
capillary .
ii.CGE uses separation based on the difference in solute size as a particle
migrate through the gel.
iii.Gels prevent the capillary walls from absorbing then solute.

Applications:
•Genetic Analysis.
•Analysis of Pharmaceuticals.
•Pharmaceuticals with Chiral Centers (Enantiomers).
•Counter-ion analysis in drug discovery.
•Protein Characterisation.

What is DNA Microarray?
• DNA microarray is a set of DNA sequences representing the entire set
of genes of an organism, arranged in a grid pattern for use in genetic
testing.
• DNA microarray is a laboratory tool used to detect the expression of
thousands of genes at the same time.

DNA Chip Technology:
a) A DNA chip can be
manufactured to contain
hundreds of thousands of
synthetic single- stranded
DNA sequences.
b) Unknown DNA from a
patient is separated into
single strands, enzymatically
cut and labeled with a
fluorescent dye.

c) The unknown DNA is inserted
into the chip and allowed to
hybridize with the DNA on the
chip.
d) The tagged DNA will bind only to the
complementary DNA on the chip. The
bound DNA will be detected by its
fluorescent dye and analyzed by a
computer. The red light is a gene
expressed in normal cells; green is a
mutated gene expressed in tumor cells;
and yellow, in both cells.
Fig: DNA Chip Technology

1. Sample
preparation
2. Purification

3. Reverse
Transcription
4. Labelling

5.Hibridization
6.Scanning
7.Normalization
and analysis

• Provides data for thousands of genes.
• One experiment instead of many.
• Fast and easy to obtain results.
• Huge step closer to discovering cures for diseases and cancer.
• Different parts of DNA can be used to study geneexpresion.
Advantages
•The biggest disadvantage of DNA chips is that they are expensive to
create.
•The production of too many results at a time requires long time for
analysis, which is quite complex in nature.
•The DNA chips do not have very long shelf life, which proves to be
another major disadvantage of the technology.
Disadvantages:

Application of DNA Microarray:
• Gene expression analysis.
• Disease diagnosis.
• Drug discovery.
• Toxicological research.
• Nutrigenomic research.

Definition Mass Spectrometry
 Mass spectrometry (MS) is an analytical technique that
ionizes chemical species and sorts the ions based on their mass-to-
charge ratio. A mass spectrum measures the molecular mass/molecular
weight of the molecule.
 A mass spectrum is a plot of relative abundance or concentration of
gaseous ion against the mass to charge ratio (m/z value)

Principle of Mass Spectrometry
 MS can differentiate different molecule according to their mass to
charge ratio of the particular molecule.
 It does not deal with the total molecule means the molecule will not be
leave as it is. The molecule will be fragmentized in different regions.
Thus, it takes mass by charge ratio of different segments of molecule
to find their identity.
 It identify atoms or molecules by comparison the spectra of unknown
molecules or atoms with known compound.
O = 16
N = 14Atoms

Components of Mass Spectrometry
The instrument consists of three major components:
• Ion Source: For producing gaseous ions from the substance being
studied.
• Analyzer: For resolving the ions into their characteristics mass
components according to their mass-to-charge ratio.
• Detector System: For detecting the ions and recording the relative
abundance of each of the resolved ionic species.

Mass Spectrometry Flow Chart
Inlet
Sample
Ionization
MS/Mass
Analyzer Detector
Data
System
Sample
preparation
2D gel
electrophoresisProtein
Extraction
Protein
digestion
Prpteases (trypsin)
Peptide
separation
Peptide separation
by HPLC & Ion
exchange
Chromatography
Eluent
Electrospray
ionization
MALDI
Quadrupole
Time-of-flight (TOF)
Quadrupole ion trap
FTICR
Microchannel plate
detectors
Electron multipliers
Faraday cups
Ion-to-photon
detectors
Mascot database
Peptide search
Sequest
High Vacuum SystemSolid
Liquid
Gas

ION SOURCE
• Since the mass analyzer utilizes only gaseous ions i.e., starting
point of mass spectrometric analysis is formation of gaseous
analyte ions.
• Non –Volatile solids are first converted in to gases and from the
gaseous sample the ions are produced in a Box like enclosure
called Ion Source.
Function
• Produces ion without mass discrimination sample.
• Accelerates ions into the mass analyzer.

Catogories of Ion sources
Gas Phase Sources
• Electron Impact Ionization (EI)
• Chemical Ionization (CI)
• Field Ionizations (FI)
Desorption Sources
• Field Desorption (FD)
• Electrospray Ionization (ESI)
• Matrix assisted desorption/Ionisation (MALDI)
• Plasma desorption (PD)
• Fast Atom Bombardment (FAB)
• Thermospray Ionization (TS)
• Secondary Ion Mass Spectrometry (SIMS)

Applications of Mass Spectrometry
 Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical

What is NMR?
• Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical
chemistry technique used in quality control and research for
determining the content and purity of a sample as well as its molecular
structure.

Working Principle of NMR
The principle of NMR usually involves two sequential steps:
1. Polarization of the magnetic nuclear spins in an applied constant magnetic
field (H0), and
2. The perturbation of this alignment of the nuclear spins by employing an
electromagnetic pulse usually radio frequency pulse.
 A computer controls the apparatus that feeds a signal to a pulse generator.
It selects pulses of high frequency radiation generated by a RF generator
that are passed to a RF amplifier and then to the coils of the sample probe.
 The Absorbed energy provides a signal which is amplified and passed to
the detector, where the RF signal is heterodyned with the original RF
source frequency and the frequency difference is amplified by an AF amplifier.
 An analogue digitize the output from the AF amplifier to digital converter
and the digitise signal is processes by a computer.

NMR Instrumentation
 Sample holder
 Permanent magnet
 Magnetic coil
 Sweep generator
 Radio frequency transmitter
 Radio frequency receiver
 Read out systems.

Advantages of NMR:
• Very sensitive to weak interaction.
• Reveal the portion of molecule involved in interaction.
• With the suitable computer apparatus we can calculate the whole 3D
structure of protein .
• Painless experiment.
Disadvantages of NMR:
• Not for the availability of higher molecular weight.
• The resolving power of the NMR is less than some other type of
experiment.
• Require high concentration of soluble protein.
• Tendency for spins to align field is weak.

Application of NMR Spectroscopy
In Structural Biology:
1. Structure determination through homogeneous compound.
2. Production of recombinant protein labelled with radioactive isotopes.
 In Medicine:
1.MRI for medical diagnosis.
2.MR microscopy in research.
3.Biochemical information from living tissue.
4.Metablomics.

Application of NMR Spectroscopy
In Chemistry:
1.Determination of structure of compounds.
2.Identification of any compound.
 In non-destructive testing:
1. Through static magnetic fields.
2. Biological sample could be studied.
3. Testing of harmful compound.

Definition of UV spectroscopy
• UV spectroscopy is an important tool in analytical
chemistry.
• The other name of UV spectroscopy is Electronic
spectroscopy as it involves the promotion of the electrons
from the ground state to the higher energy state.

Principle of UV Spectroscopy
• Spectroscopy is related to the interaction of light with matter.
• As light is absorbed by matter, the result is an increase in the energy
content of the molecules.
• When ultraviolet radiations are absorbed, this results in the excitation
of the electrons from the ground state towards a higher energy state.
• Molecules containing non bonding electrons(n electrons) can absorb
energy in the form of ultraviolet light to excite these electrons to
higher anti bonding molecular orbitals.
• The more easily excited the electrons, the longer the wavelength of
light it can absorb.
• The absorption of ultraviolet light by a chemical compound will
produce a distinct spectrum which aids in the identification of the
compound.

Instrumentation of UV spectroscopy
1. Light source
• Tungsten filament lamps and Hydrogen-Deuterium lamps are
used and suitable light source as they cover the whole UV
region.
• Tungsten filament lamps are rich in red radiations; more
specifically they emit the intensity of Hydrogen-Deuterium
lamps falls below 375 nm.

Instrumentation of UV spectroscopy cont..
2. Monochromator
• Monochromators generally is composed of prisms and slits.
• Most of the spectrophotometers are double beam spectrophotometers.
• The radiation emitted from the primary source is dispersed with the
help of rotating prisms.
• The various wavelengths of the light source which are separated by the
prism results in a series of continuously increasing wavelength to pass
through the slits for recording purpose.
• The beam selected by the slit is monochromatic and further divided
into two beams with the help of another prism.

3. Sample and reference cells
• One of the two divided beams is passed through the sample solution
and second beam is pass through the reference solution.
• Both sample and reference solution are contained in the cells.
• These cells are made of either silica or quartz. Glass can not be used
for the cells as it also absorbs light in the UV region.
4. Detector
• Generally two photocells serve the purpose of detector in UV
spectroscopy.
• One of the photocell receives the beam from the reference.
• The intensity of the radiation from the reference cell is stronger than
the beam of sample cell.

5. Amplifier
• The alternating current generated in the photocells is transferred to the
amplifier.
• The amplifier is coupled to a small servometer.
• Generally current generated in the photocells is of very low intensity,
the main purpose of amplifier is to amplify the signals many times so
we can get clear and recordable signals.
6. Recording devices
• Most of the time amplifier is coupled to a pen recorder which is
connected to the computer.
• Computer stores all the data generated and produces the spectrum of
the desired compound.

Applications of UV Spectroscopy
1.Detection of Impurities
• UV absorption spectroscopy is one of the best methods for
determination of impurities in organic molecules.
• Additional peaks can be observed due to impurities in the sample and
it can be compared with that of standard raw material.
• By also measuring the absorbance at specific wavelength, the
impurities can be detected.
2. Detection of Functional Groups
• This technique is used to detect the presence or absence of functional
group in the compound.
• Absence of a band at particular wavelength regarded as a evidence for
absence of particular group.

Applications of UV Spectroscopy Cont…
3. Identification of an unknown compound
• An unknown compound can be identified with the help of UV spectroscopy.
• The spectrum of unknown compound is compared with the spectrum of a reference
compound and
• If both the spectrums coincide then it confirms the identification of unknown
substance.
4.Structure elucidation of organic compounds
• UV spectroscopy is useful in the structure elucidation of organic molecules.
• From the location of peaks and combination of peaks, it can be concluded that
whether the compound is saturated or unsaturated, hetero atoms are present or not.

Applications of UV Spectroscopy Cont…
5.Chemical kinetics
• Kinetics of reaction can also be studied using UV spectroscopy.
• The UV radiation is passed through the reaction cell and the
absorbance changes can be observed.
6.Qualitative analysis
• UV absorption spectroscopy can characterize those types of
compounds which absorbs UV radiation.
• Identification is done by comparing the absorption spectrum with the
spectra of known compounds.

Definition of Infrared Spectroscopy
• IR spectroscopy deals with the infrared region of the
electromagnetic spectrum, i.e. light having a longer
wavelength and a lower frequency than visible light.
• Infrared Spectroscopy generally refers to the
analysis of the interaction of a molecule with
infrared light.
• The IR spectroscopy concept can generally be
analyzed in three ways: by measuring reflection,
emission, and absorption.

Principle of Infrared Spectroscopy
• The IR spectroscopy theory utilizes the concept that molecules tend
to absorb specific frequencies of light that are characteristic of the
corresponding structure of the molecules.
• The energies are reliant on the shape of the molecular surfaces, the
associated vibronic coupling, and the mass corresponding to the
atoms.
• For instance, the molecule can absorb the energy contained in the
incident light and the result is a faster rotation or a more
pronounced vibration.

IR Spectroscopy Instrumentation

Advantages of IR
• Qualitative and quantitative analysis: One of the key advantages of
Infrared spectroscopy is without destroying the sample it can provide
qualitative and quantitative chemical information.
• Sample Preparation: The major advantage of infrared spectroscopy is
that the sample does not need any particular preparation.
• Sensitive and Time-saving technique: IR spectroscopy is very sensitive,
hence it required minimum sample quantity to scan the sample spectrum
and it takes a few seconds to scan a whole range of IR.
• It's versatility: Solid, liquid, gases and semisolid samples can be
analyzed by the IR spectroscopy.
• Easy for interpretation: The Peak intensities, peak positions, peak
widths, shapes, and functional groups provide all helpful information.

Disadvantages of IR
• Disadvantages include sometimes difficult handling procedures and
maintenance of the sample cells.
• There are no infrared spectra in atoms or monatomic ions, hence it
cannot analyses.
• To use infrared spectroscopy is that it requires very sensitive and
properly tuned devices.
• The sample having aqueous solutions and complex mixtures are
complicated to analyze by infrared spectroscopy.

Applications of IR
• It is used extensively in forensic science ( eg, To analyse paint
fragments from vehicles in hit and run offences )
• Monitoring the degree of unsaturation in polymers.
• Quality control in perfume manufacture.
• Drug analysis.
• Testing the breath of suspected drunken drivers for ethanol.

DNA and Protein Analysis Methods Presentation

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