Bioanalytical and Biophysical

Techniques
MBTE11

Dr. Sravanti
UNIT-I
 DNA sequencing-
 Principle & technique of Pyrosequencing,
 Introduction to Next generation sequencing.
 Protein sequencing: Edman degradation,
 Separation techniques: Capillary electrophoresis,
 2D- Gel Electrophoresis,
 Chromatographic technique- High Performance
Liquid Chromatography, Reverse Phase-HPLC.
 Centrifugation- Preparative centrifugation,
Analytical Ultra centrifugation,
 Flow cytometry- Fluorescence activated cell
sorting (FACs) and its applications.
 Tests &Marks
 Internal- 30 Marks
 10 Marks- Assignment/Test/Problems to solve
 7 Marks- Discussion on a topic
 3 Marks- General Class performance
 Topics for discussion
1. Pacific Biosystem NGS
2. Solexa NGS
3. DNA chip sequencing
4. FRET
5. Human Genome project
6. Fluorescence spectroscopy
7. Electron Microscopy
8. Scanning electron microscopy SEM
9. EPR- electron paramagnetic resonance
10. X-ray powder diffraction
11. SAXS- small angle X-ray scattering
12. Neutron Diffraction
13. ITC paper
14. DSC paper
15. SPR paper
16. DNA microarray fabrication techniques
17. Protein microarray
Topic discussion
 3-4 page write up (minimum)
 History if any
 Exact Protocol
 Principle, Instrumentation
 Discussion of the application in a recently
published article (2011 above)
 References
 The paper that is discussed
 Books

 Principles of Proteomics- R. M. Twyman- Library

 Biophysics- Vasantha Pattabhi, N. Gautham- Buy

 Principles and Techniques of Biochemistry and Molecular

Biology- Keith Wilson, John Walker- Library

 Physical Biochemistry- David Freifelder-

Library/Reference
Understand and present in this way
 Principle
 Instrumentation
 Technique/Methodology
 Applications
 Limitations/Advantages/Disadvantages
Pyrosequencing
Note: No actual houses
are burned down in
pyrosequencing
 Pyrosequencing
 A novel DNA sequencing method in
which addition of a nucleotide to the end
of a growing polynucleotide is detected
directly by conversion of the released
pyrophosphate into a flash of
chemiluminescence.
Pyrosequencing
(Life Sciences / Roche 454)
 A luciferase is an enzyme which emits light
in the presence of ATP.

Several organisms, such as the American firefly and the

poisonous Jack-o-lantern mushroom, produce luciferases.
Detecting polymerase activity

 Recall: Pyrophosphate is also known as PPi, also

known as “two phosphate groups stuck
together”. During replication, each addition of a
dNTP releases pyrophosphate.

 In the reaction mixture,

◦ PPi allows adenosine phosphosulfate (APS) to be
converted to ATP;
◦ this ATP allows luciferase to oxyluciferase (emit light).

 Measures strand extension as it happens

Equations
Pyrosequencing cycle- read out
 Add dATP. If light is emitted, your sequence
starts with A. If not, the dATP is degraded
(or elutes past immobilized primer).
 Add dGTP. If light is emitted, the next base
must be a G.
 Then add T, then C. You now know at least
one (maybe more) base of the sequence.
 Repeat!
 Order of addition is very important
 Order should not be changed during process
The strand synthesis reaction is carried out in
the absence of dideoxynucleotides.
Each dNTP is added individually, along with a
nucleotidase enzyme that degrades the dNTP if
it is not incorporated into the strand being
synthesized. Incorporation of a nucleotide is
detected by a flash of chemiluminescence
induced by the pyrophosphate released from
the dNTP. The order in which nucleotides are
added to the growing strand can therefore be
followed.
 Pyrosequencing output

Runs of bases produce higher peaks – for instance, the sequence for (a)
is GGCCCTTG. Sample (c) comes from a heterozygous individual (hence
the heights in multiples of ½)
Advantages
 Direct read out during incorporation of
nucleotides
 Extra step of separation and detection
not required
 Labeling of test DNA not required
 Easily automated
 Next-Generation Sequencing NGS

 In 1977 the first genome, that of the 5,386

nucleotide (nt), single-stranded bacteriophage
φX174, was completely sequenced using a
technology invented just a few years earlier

 In the late 1980s, semi-automated sequencers

with higher throughput became available still only
able to determine a few sequences at a time
Breakthroughs in the field
A breakthrough in the early 1990s was the
development of capillary array electrophoresis
and appropriate detection systems .

As recently as 1996, these developments converged

in the production of a commercial single capillary
sequencer (ABI Prism 310).

In 1998, the GE Healthcare MegaBACE 1000 and

the ABI Prism 3700 DNA Analyzer became the
first commercial 96 capillary sequencers, a
development which was termed high-throughput
sequencing.
Discoveries which revolutionized
sequencing technique
Improvements in the rate of DNA
sequencing over the past 30 years and into the future.

MR Stratton et al. Nature 458, 719-724 (2009) doi:10.1038/nature07943

 Principles for different NGS
 Sanger capillary sequencing-Capillary sequencing
 Roche/454 GS FLX Titanium sequencer-
Pyrosequencing (2005)
 Illumina Genome Analyzer (Solexa) -Reversible
terminator chemistry (2007)
 Applied Biosystems SOLiD-Sequencing-by-ligation
 Helicos HeliScope-Virtual terminator chemistry
 Ion Torrent (2010)-pH based
Comparison of different NGS
Steps in NGS system
 Polony PCR

 Polony sequencing is an inexpensive

 but highly accurate multiplex sequencing technique that can be used to
“read” millions of immobilized DNA sequences in parallel.

 This technique was first developed by Dr. George Church's group at

Harvard Medical School

 His 1984 Harvard PhD included the first methods for direct genome
sequencing, molecular multiplexing & barcoding. These led to the first
genome sequence (pathogen, Helicobacter pylori) in 1994 . His
innovations have contributed to nearly all "next generation" genome
sequencing methods and companies (CGI, Life, Illumina, nanopore). This
plus chip-based DNA synthesis and stem cell engineering resulted in
founding additional application-based companies spanning fields of
medical diagnostics
Sanger capillary sequencing
 Sanger capillary sequencing
 Input DNA is fragmented and cloned into bacterial vectors for
 in vivo amplification.

 Reverse strand synthesis is performed on the obtained copies starting from a

known priming sequence and using a mixture of deoxy-nucleotides (dNTPs)
and dideoxy-nucleotides
 (ddNTPs).

 The dNTP/ddNTP mixture randomly causes the extension

 to be non-reversibly terminated, creating differently extended molecules.

 Subsequently, after denaturation,

◦ clean up of free nucleotides,
◦ primers, and the enzyme, the resulting molecules are sorted
◦ using capillary electrophoresis by their molecular weight (corresponding to the
point of termination) and the
◦ fluorescent label attached to the terminating ddNTPs is read out sequentially.
Features of SCS
 384 sequences of 600-1000 nt length can
be sequenced in parallel

 96-capillary instruments yield

 6MB DNA sequence/day
 $500/1MB
 Error rate is low- 1/10,000 -1/100,000
Roche/454 GS FLX Titanium sequencer

 The 454 sequencing platform was the first of the

new high-throughput sequencing platforms on
the market (released in October 2005).

 It is based on the pyrosequencing approach

developed by Pa°l Nyre´n and Mostafa Ronaghi
at the Royal Institute of Technology, Stockholm in
1996 .
Steps involved
Mechanism of amplification-
Emulsion PCR
 In contrast to the Sanger technology, pyrosequencing is
based on
◦ iteratively complementing single strands
◦ and simultaneously reading out the signal emitted from the
nucleotide being incorporated
◦ also called sequencing by synthesis, sequencing during
extension.

 Electrophoresis is therefore no longer required to

generate an ordered read out of the nucleotides, as the
read out is now done simultaneously with the sequence
extension.
 In 2005, pyrosequencing technology was
parallelized on a picotiter plate by 454 Life
Sciences (later bought by Roche Diagnostics)
to allow high-throughput Sequencing.

 The sequencing plate has about two million

wells – each of them able to accommodate
exactly one 28-mm diameter bead covered
with single-stranded copies of the sequence to
be determined.
 The beads are incubated with
◦ a polymerase and single-strand binding proteins and,
together with
◦ smaller beads carrying the ATP sulfurylases
◦ and luciferases, gravitationally deposited in the wells.

 Free nucleotides are then washed over the flow

cell and the light emitted during the
incorporation is captured for all wells in parallel

 high-resolution charge-coupled device (CCD)

camera
 One of the main prerequisites for applying this array-based
pyrosequencing approach is covering individual beads with
multiple copies of the same molecule.

 This is done by first creating sequencing libraries in which

every individual molecule gets two different adapter
sequences, one at the 50 end and one at the 30 end of the
molecule.

 In the case of the 454/Roche sequencing library

preparation, this is done by sequential ligation of two pre-
synthesized oligos.

 One of the adapters added is complementary to

oligonucleotides on the sequencing beads and thus allows
molecules to be bound to the beads by hybridization.
Features of Roche/454 GS
Illumina Genome Analyzer
 Illumina Genome Analyzer
 Reversible terminator chemistry applied by the
Illumina GA.

 Sequencing primers are annealed to the adapters of

the sequences to be determined.

 Polymerases are used to extend the sequencing

primers by incorporation of fluorescently labeled
and terminated nucleotides.

 The incorporation stops immediately after the first

nucleotide due to the terminators.
 The polymerases and free nucleotides are
washed away

 The label of the bases incorporated for each

sequence is read with four images taken through
different filters using two different lasers (red: A,
C and green: G, T) to illuminate fluorophores.

 Subsequently, the fluorophores and terminators

are removed and the sequencing continued with
the incorporation of the next base.

◦ Fluorophore attached by Photocleavable linker

◦ Capping of 3′-OH
 Sample library generation- In vitro
 Millions of different DNA templates are
immobilized on the surface of a chip

 3' 5' adaptors are ligated

 Solid-phase bridge amplification

Protein sequencing
 Frederic Sanger first time achieved
complete sequence of protein (bovine
insulin) in 1953. For his work, he was
awarded the Nobel Prize of Chemistry in
(1958).

 Why is it important to sequence

Proteins??
◦ Learn
 There are several applications of protein
sequencing, which are:-
 a) Identification of the protein family to which a
particular protein belongs and finding the
evolutionary history of that protein. Function
prediction.
 b) Prediction of the cellular localization of the
protein based on its target sequence (sequence of
amino acids at the N terminal end of the protein
which determines the location of the protein
inside the cell).
 c) Prediction of the sequence of the gene
encoding the particular protein.
 d) Discovering the structure and function of a
protein through various computational methods
and experimental methods.
 Two main methods include
 Edman degradation
 Any other method??
 Mass Spectrometry
 Protein sequence can also be generated
from the DNA/mRNA sequence that
codes for the protein
Proteins- peptide bonds, amino acids
Protein Sequencing- Preliminary steps
 For multisubunit proteins, the individual protein chains must
first be separated

 Break interchain disulfide bonds, if necessary

 Two reagents are commonly used:
◦ performic acid
◦ Mercaptoethanol

 After breaking disulfide bonds, the chains are separated by

disrupting noncovalent interchain interactions with pH
extremes, 8 M urea, 6 M guanidium hydrochloride, or high salt

 Then the individual protein chains are separated by

electrophoresis or chromatography on the basis of size or
charge
 Sequencing
 Once each protein is purified the amino acid
sequence is determined by:
 1) determining the amino acid composition (how
many of each amino acid are in the protein)

 2) identifying the amino and carboxyl terminal

amino acids

 3) cleaving the protein into two or more sets of

peptides using specific enzymatic or chemical
reagents such as trypsin or cyanogen bromide
 4) determining the amino acid sequence
of each of the peptide fragments (N
terminal or C-terminal)

 5) determining the entire protein

sequence from the sequences of
overlapping peptide fragments

 6) locating the position of disulfide

bridges between cysteines
Peptide sequencing
•Pehr Edman, a Swedish
Biochemist working at St.
Vincent’s School of Medical
Research in Australia, develops a
method for determining amino
acid sequences in proteins 1n
1965

•Commonly known as the

‘Edman degradation,’ the method
later becomes the basis for
automated protein sequencing

•N-terminal sequencing
 Edman degradation

◦ Edman reagent-Phenylisothiocyanate or PITC adds to

N-terminus, then acid treatment cleaves the N-
terminal amino acid as a PTH derivative

◦ Identified by extraction, treatment with aqueous acid

and analysis by chromatography

◦ The remaining protein chain is intact and the cycle

can be repeated

◦ Under ideal conditions the sequence of 30-60 amino

acids can be determined
 The Edman chemistry cycle consists of three stages:

1. Coupling
 The N-terminus of the protein couples with PITC under basic conditions to form
a phenylthiocarbamyl (PTC)-polypeptide.

2. Cleavage
 The peptide bond of the N-terminal PTC-residue undergoes acid cleavage from
the polypeptide chain.
 This results in the release of an unstable anilinothiazolinone (ATZ) derivative of
the amino acid.

3. Conversion
 The unstable ATZ-amino acid is converted into the corresponding
phenylthiohydantoin (PTH) derivative.
 The PTH-amino acid is stable.

 Identification of amino acid:

 At the end of each cycle of Edman degradation, the PTH-amino acid is separated
from reaction by-products and identified, by HPLC chromatography and UV
absorbance.
Chemistry involved
Phenylisothiocynate (PITC) Phenylthiocarbamyl, (PTC)-polypeptide

Coupling

Conversio
n

Cleavage

Anilinothiazolinone (ATZ)
Automated Edman- 1982
Applied Biosystems, Inc. releases its
first product, the Model 470A
protein sequencer.

The machine vastly improves the

sensitivity of the ‘Edman degradation’
used to determine the order of
amino acids in protein molecules, and
significantly expands the utility of
protein chemistry in industrial and
biomedical applications.

Leroy Hood and Michael Hunkapiller

had invented the sequencer at Cal
Tech in the late 1970s, but no
instrument company was willing to
develop it.

Applied Biosystems was founded in

1981 by venture capitalist William K.
Bowes and former Hewlett-
Packard engineers Sam Eletr and
André Marion, in order to
commercialize the invention -