The document discusses DNA, RNA, gene expression, and various techniques related to studying nucleic acids including microarray, PCR, and real-time PCR. It defines the key components and structures of DNA and RNA, describes the central dogma of molecular biology regarding gene expression through replication, transcription and translation, and provides details on experimental procedures like microarray, PCR cycling, and real-time quantitative PCR using different fluorescent probes.

1. By
Priyanka Sharma
Research Scholar (Pharmacology)
DELHI PHARMACEUTICAL SCIENCES AND RESEARCH
UNIVERSITY
Study Of Microarray

2. DNA
 Deoxyribonucleic acid
 DNA - a polymer of deoxyribo-
nucleotides.
 Usually double stranded.
 And have double-helix structure.
 found in chromosomes,
mitochondria and chloroplasts.
 It acts as the genetic material in
most of the organisms.
 Carries the genetic information

3.  DNA Structure
 DNA structure is often divided into four
different levels primary, secondary,
tertiary and quaternary.
DNA has three main components
1. Deoxyribose (a pentose sugar)
2. Base (there are four different ones)
3. Phosphate

4.  Nucleotide Structure
 Nucleotides are formed by the condensation of a sugar,
phosphate and one of the 4 bases
 The following illustration represents one nucleotide
HH
H
O
CH2
Base
Deoxyribose
5′
4′
3′
OH
O
O P O
O–
1′
H
2′
Phosphate H
Nucleotide

5. RNA
 Ribonucleic Acid
 RNA is a polymer of
ribonucleotides linked together by
phosphodiester linkage.
 RNA was first genetic material.
 Usually single stranded and helical
in structure.
 But double stranded also present
in some viruses

6. RNA structure
 There are also three main component
a) Phosphate Group
 Nucleotide
O
CH2
Base
Phosphate
5′
4′
H
1′
H
3′
OH
Ribose
2′
OH
b) Sugar(Ribose)
c) And Nitrogenous base
O
O P O
O–
HH
A, G, C or U
RNA Nucleotide

7. GENE EXPRESSION
GENE EXPRESSION
It is the process by which a gene's DNA sequence is
converted into the structures and functions of a cell.
Non-protein coding genes are not translated into
protein.
Genetic information, chemically determined by DNA
structure is transferred to daughter cells by DNA
replication and expressed by Transcription followed
by Translation.

8. Gene expression is a multi-step process which
involves
Replication Transcription Translation
Replication:
 It is a process in which DNA copies itself to produce identical
daughter molecules of DNA.
 As the newly synthesized DNA has one half of the parental DNA
and one half of new DNA.
STEPS INVOLVED IN REPLICATION
Initiation Elongation Termination

9. • Initiation
– involves assembly of replication fork (bubble) at origin of
replication
• sequence of DNA found at a specific site
• Elongation
– Parental strands unwind and daughter strands are synthesized.
– the addition of bases by proteins
• Termination:
– the duplicated chromosomes separate from each other. Now,
there are two IDENTICAL copies of DNA.
Segments of single-stranded DNA are called template strands.
Copied strand is called the complement strand
• LEADING STRAND: synthesized 5’ to 3’ in the direction of thereplication
fork movement.
-continuous requires a single RNA primer
• LAGGING STRAND: synthesized 5’ to 3’ in the opposite direction.
– semidiscontinuous requires many RNA primers , DNA is synthesized in short
fragments.
DNA Replication Steps:

11. Experimental Procedure Flow Chart
RNA (250 ug)
cDNA
cRNA + labelling Cys-dye
Purification
Quantification (Conc RNA+Dye) via Bio-analyser
SA [efficiency of labeling]
Calculate the yield [elute vol *conc]
Hybridization for 24 hr
Washing (via buffer)
Scanning (Microarray plate scanner)
Decode Image
Analysis

12. • Initiation
• Elongation
• Termination:
Two termination mechanisms are well known :- Intrinsic
termination (Rho-independent termination) & Rho-
dependent termination
Transcription Steps:
– RNA polymerase (RNAP) recognises and binds to a specific
region in the DNA called promoter
– There are two different base sequences on the coding strand
which the RNA polymerase recognises and for initiation
– RNA synthesis then proceeds with addition of ribonucleotide
ATP, GTP, CTP and UTP as building units.
– One DNA strand called the template strand serves as the
matrix for the RNA synthesis

13. • Initiation : Initiation of translation is divided into four stages:-
• Elongation: Elongation of the polypeptide chain involves addition of
amino acids to the carboxyl end of the growing chain. During elongation
the ribosome moves from the 5’ – end to the 3’ – end of the mRNA that
is being translated
• Elongation is divided into Three steps:-
• Binding of aminoacyl-tRNA to Asite
• Formation of peptide bond
• Translocation
• Termination: Termination occurs when one of the
three termination codons moves into the A site
Translation Steps: The mRNA carries genetic
information encoded as a ribonucleotide sequence from the
chromosomes to the ribosome.
– Dissociation of Ribosome
– Formation of 43s preinitiationcomplex
– Formation of 48s initiation complex
– Formation of 80s initiation complex

14. PolymeraseChainReaction
Polymerase chain reaction (PCR) is a method widely used in molecular
biology to make several copies of a specific DNA segment. Using PCR,
copies of DNA sequences are exponentially amplified to generate thousands
to millions of more copies of that particular DNA segment.
What does PCR need?
• Template (the DNA you areexploring)
• Sequence-specific primers flanking the target sequence, Forward & Reverse.
• Polymerases
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium chloride (enzyme cofactor)
• Buffer
• Water, mineral oil

15. Steps in PCR
 Denaturation 93 to 95°C 1min
 Annealing 50 to 55°C 45sec
 Elongation 70 to 75°C 1-2min

16. • Denaturation:
• Annealing:
• Elongation:
Taq polymerase binds to the template DNA and starts adding nucleotides that are
complementary to the first strand.
This happens at 72°C as it is the optimum temperature for Taq Polymerase
PCR Steps:
Denaturation is the first step in PCR, in which the DNA strands are separated by
heating to 95°C.
The Hydrogen bonds between the two strands breaks down and the two strands
separates.
Annealing is the process of allowing two sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and primers is done by cooling the DNA to
55°C.
Time taken to anneal is 45 seconds.

17. PCR Cycle
Denaturation

18. Elongation
Annealing

19.  Denaturation: 94°- 95°C
 Primer Annealing: 55°- 65°C
 Elongation of DNA: 72°
 Number of Cycles: 25-40
 No target products are made until the third
cycle.
 At 30 cycles there are 1,073,741,764 target
copies (~1×109).
PCR Cycles Review

20. Applications of PCR
 Screening human DNA samples for mutations
associated with genetic diseases such as
thalassemia and cystic fibrosis.
 Can detect the presence of viral DNA before it turns in
to a killer.
 PCR enables rapid amplification of template DNA for
screening of uncharacterized mutations
 Can obtain sequences from hair, blood stain, bones,
other forensic specimens, other remains preserved at
archaeological sites.

21. RealTime-PCR

22. What is Real Time PCR?
Real Time PCR is a technique in which fluoroprobes bind to specific target
regions of amplicons to produce fluorescence during PCR.
The fluorescence, measured in Real Time, is detected in a PCR cycler with an
inbuilt filter flurometer.
What are Fluorescent dyes?
When a population of fluorochrome molecules is excited by light of an
appropriate wavelength, fluorescent light is emitted. The light intensity can be
measured by flurometer or a pixel-by-pixel digital image of the sample.

23. Excitation and Emission: Fluorodyes absorb light at one wavelength &
thereby boosts an electron to a higher energyshell.
• The excited electron falls back to the ground state and the flurophore
re- emits light but at longer wavelength.
•This shift makes it possible to separate excitation light from emission light
with the use of optical filters.
•The wavelength (nm) where photon energy is most efficiently captured is
defined as the Absorbancemax & the wavelength (nm) where light is most
efficiently released is defined as the Emissionmax.
What is Fluorescence Resonance Energy
Transfer (FRET)?
FRET is a distance dependent interaction
between the excited states of 2 dye molecules in
which excitation is transferred from a donor
molecule to an acceptor molecule without
emission of a photon

24. Quantitating Fluorescence
A flurometer exploits the principles of fluorescence to quantitate
fluorescent (dye) molecules in the following way:
A strong light source which produces light within a specific light
range ( eg xenon arc lamp) is focused down to a tight beam.
The tight beam of light is sent through a filter which removes
most of the light outside of the target wavelength range.
The filtered light beam passes through the liquid target sample
striking some of the fluorescent molecules in the sample.
Light emitted from the fluorescent molecules travels orthogonal
to the excitation light beam pass through a secondary filter that
removes most of the light outside of the target wavelength range.
The filtered light then strikes a photodetector or
photomultiplier which allows the instrument to give a relative
measurement of the intensity of the emitted light.

27. Real Time PCR Instruments
 LightCycler (Idaho Technologies Roche)
 Rotor-Gene (Corbett Research)
 iCycler (BioRad)
 Mx4000™ Multiplex Quantitative PCR System
 ABI Prism 7700 (Perkin-Elmer-Applied-Biosystem)
 SmartCycler (Cephid)
Instruments

28. Real Time Detection
1a. Excitation filters
1b. Emission filters
Tungsten halogen light source
(350 - 1000nm continuous)
Microplate format
Cycler
iCycler from BioRad

29. Probe types & Design
dsDNA Binding Dye
 SYBR Green I
 SYBR Green II
 EVAGreen
 LC Green
 BEBO
 YO-PRO
 SYTO family

30. Sybr Green PCR Assay
Stronger signal
Higher selectivity for dsDNA
Lesser sequence dependent
Higher stability
Lesser inhibitory for Taq
Higher resolution in melting curves
Less hazardous and mutagenicity)
Binds to
Non specific PCR product
Primer dimer

31. Hydrolysis Probes (TaqMan)
When intact, the fluorescence of the reporter
When intact, the fluorescence of the reporter
Quencher
Probe hybridizes to the target
dsDNA-specific 5'—>3' exonuclease activity of Taq or Tth cleaves
off the reporter
 Reporter is separated from the quencher.
 Fluorescent signal
Signal is proportional to the amount of
amplified product in the sample

33. Hairpin probes: Molecular beacons
Molecular Beacons are hairpin structures composed of a (25–40 nt) nucleotide
base paired stem and a target specific nucleotide loop.
The loop consists of target specific nucleotide (probe) sequences (15–30 nt)
A fluorescent moiety (reporter)is attached to 5’ end and a quencher moiety is
attached to 3’end. The stem keeps both the moieties in close proximity so that
fluorescence is quenched.
Loop
Stem

34. Denaturation
Extension
5’
Q
5’ 3’
5’
5’
3’
3’
5’
5’3’
3’5’
5’
Primer molecular
Beacon annealing
3’5’
5’
5’
3’
5’
QR
5’
3’
Operation of Molecular Beacon
(MB): MB is non-fluorescent due to
fluorescent quencher (Q) and
close proximity of the non-
the
fluorescent Reporter
The probe denatures and the loop
anneals to the target sequence of
the amplicon
Separating the quencher from the
fluorophore and thereby producing
fluorescence which is proportional to
the amplicons produced during PCR
MB is displaced not destroyed
during amplification, because a DNA
polymerase lacking 5' exonuclease
activity is used

36. 3’ Quencher
Blocker
5’ Reporter
Complementary sequence
Scorpion primer consists of:
PCR
primer
The loop of the Scorpions probe includes a sequence that is complementary to
an internal portion of the sequence it primes.
During the first amplification cycle, the Scorpions primer is extended, and the
sequence complementary to the loop sequence is generated.
After subsequent denaturation and annealing, the loop of the Scorpions probe
hybridizes to the internal target sequence, and the reporter is separated from the
quencher. The resulting fluorescent signal is proportional to the amount of
amplified product in the sample.
The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent
read-through during the extension of the opposite strand.
Scorpion Primers

37. The primer is
part of the
Scorpion probe
The primer is
extended
The template &
probe denature
The primer binds
to the target
Scorpion stem-loop
format
Primer, stopper to
read PCR through,
prevent
probe
sequence, fluorophore &
quencher (detection system).
The probe binds to the
complimentary sequence
of the DNA

38. Hybridization Probes
These assays use two sequence-specific oligonucleotide probes in
addition to two sequence specific primers. The two probes are
designed to bind to adjacent sequences in the target. The probes
are labeled with a pair of dyes that can engage in FRET. The
donor dye is attached to the 3' end of the first probe, while the
acceptor dye is attached to the 5' end of the second probe.
During real-time PCR, excitation is performed at a wavelength
specific to the donor dye, and the reaction is monitored at the
emission wavelength of the acceptor dye. At the annealing step,
the probes hybridize to their target sequences in a head-to-tail
arrangement. This brings the donor and acceptor dyes into
proximity, allowing FRET to occur.
The increase in PCR product is proportional to amount of
fluorescence

39. Probe 2
Probes hybridize to their
target sequences in a
head-to-tail arrangement. FRET
Probe 1
Hybridization Probe

40. Hybridization probes
h
D
A
h
A
FRET
D
Amplicon
Probe 1
Probe 2
D FAM A LC red 640

41. SUNRISE UNIPRIMER PROBE
Similar to Molecular Beacon except the stem contains a poly A (15 mer)
tail. This tail is complimenatry to thepolyT tail of one f the primers.
PolyA Tail
Q

42. Q
R
Q R
hv
Primer with polyT tail
Sunrise Probe with polyA tail
binds to the primer polyT tail at
annealing.
AAAAAAAAAAAAAA
The Sunrise probe changes conformation during
denaturation & quenching by DABCYL is removed
allowing FITC to fluoresce
Sunrise UniPrimer Probe is a modification of Molecular Beacon

43. Commonly Used Fluorescent Probes
Detection Chemistries
2%
3%
9%
9%
15%
19%
78%
0% 10% 20% 30% 40% 50% 60% 70% 80%
TaqMan probes
Molecular Beacons
FRET probes
LUX fluorogenic
primers
MGB Eclipse probes
Other
Scorpion probes

44. •Amplification plot is the plot of
fluorescence signal versus cycle
number.
•Initial cycles of PCR, there is little
change in fluorescence signal. This
defines the baseline of
amplification plot.
•An increase in fluorescence above
the baseline indicates detection of
accumulated PCR product.
The parameter CT(Threshold cycle) is defined as the fractional
cycle number at which the fluorescence passes the fixed threshold.
Real-Time PCR Terminology

45. During the exponential phase, none of the reaction components is limiting; as
a result, CT values are very reproducible for reactions with the same starting
copy number.
On the other hand, the amount of PCR product observed at the end of the
reaction is very sensitive to slight variations in reaction components.
Effect Of Limiting Reagent

46. •The Threshold line is the level
of detection or the point at which
a reaction reaches a fluorescent
intensity above background.
•The threshold line is set in the
Threshold cycle CT
exponential phase of the
amplification for the most
accurate reading.
•The cycle at which the sample
reaches this level is called the
Cycle Threshold, CT.
•CT value of 40 or more means no amplification and cannot
be included in the calculations.
•A sample whose Ct is 3 cycles earlier than another's has
23 = 8 times moretemplate.

47. •Rn+ is the Rn value of a reaction containing all components (the
sample of interest)
• Rn- is the Rn value detected in NTC (baseline value)
•Rn is the difference between Rn+ and Rn-. It is an indicator of
the magnitude of the signal generated by the PCR
• Rn is plotted against cycle numbers to produce the
amplification curves and to estimate the CT values

48. REALTIME-PCR APPLICATIONS
Application in Molecular Diagnostics
 Clinical microbiology and Food microbiology
 Gene expression
 viral quantitation
 Single Nucleotide Polymorphism (SNP) analysis
 Clinical oncology
 Cancer
 Analysis of cellular immune response in peripheral blood
 Chromosome aberrations

49. Detection of Pathogens
A Scorpion Probe Based Real-Time PCR Assay for Detection of E. coli O157:H7 in
Dairy Products.
RTi-PCR method based on Scorpion probe targeting the
eae gene of E. coli O157:H7.
Genomic DNA isolation
Primer-probes were designed
based on eaeA gene sequences
standard curve preparation of 10-
fold serial dilution
Sensitive -2log CFU/mL
Singh et al.,2009

50. SNP Genotyping
Multiplex TaqMan assay for SNP genotyping

51. SNP Identification Molecular Beacons

52. High Resolution Melting
Mutations in PCR products are detectable by HRM analysis because they
lead to changes in DNA melting curves.
The A:T to G:C interchange, which is the most common SNP results in a
difference of about 1 °C in Tm, which is readily detected by HRM.
Dyes used for HRM analysis:
•SYTOR 9 dye (Invitrogen)
•LCGreenR, LCGreenR Plus+ (Idaho Technologies)
•EvaGreen™ dye (Biotium Inc.)
•SYBRR GreenER™ dye (Invitrogen)

53. DNA Methylation Analysis
Methylation influence gene expression by affecting the interactions with DNA
of both chromatin proteins and specific transcription factors.
Bisulfite treatment converts cytosine to uracil while
5-methy cytosine is resistant to the conversion.
Methylated DNA having “C” will have a higher melting temperature than
unmethylated DNA having “T” at same position.
This can be detected by melt
curve analysis.

54. DNA-Microarray
The large scale genome squncing effort and the ability to
immobilize thousands of DNA fragments on coated glass slide or
membrane, have led to the development of microarray technology.
A microarray is a pattern of ssDNA probes which are
immobilized on a surface called a chip or a slide.
Microarrays use hybridization to detect a specific DNA or RNA
in a sample.
DNA microarray uses a million different probes, fixed on a solid
surface.

55. An array is an
orderly arrangement of
samples where matching of
known and unknown DNA
samples is done based on base
pairing rules.
An array experiment makes use
of common assay systems such
as microplates or standard
blotting membranes.
Fig-01 Robotic arm with
spotting slides

56. The core principle behind
microarrays is hybridization
between two DNAstrands.
 Fluorescent labeled target
sequences that bind to a probe
sequence generate a signal that
depends on the strength of the
hybridization determined by
the number of paired bases.
Fig-02 Array hybridization

57.  DNA microarray technology may be defined as a
high-throughput and versatile technology used for
parallel gene expression analysis for thousands of
genes of known and unknown functions.
 Used for detection of polymorphisms and mutations
in genomic DNA
 A DNA microarray is a collection of microscopic
DNA spots on solid surface. Each spot contains
picomoles of a specific DNA sequence, known as
probes or reporters.

58.  Each identified sequenced gene on the glass, silicon
chips or nylon membrane corresponds to a fragment
of genomic DNA, cDNAs, PCR products or
chemically synthesized oligonucleotides of up to
70mers and represents a single gene.
 Probe-target hybridization is usually detected and
quantified by detection of fluorophore, silver, or
chemiluminescence labeled targets to determine
relative abundance of nucleic acid sequences in the
target.

60.  The principle of DNA microarray technology is
based on the fact that complementary sequences
of DNA can be used to hybridise, immobilised
DNA molecules.
 There are four major steps in performing a
typical microarray experiment.
Sample preparation
and
labeling
Hybridisation Washing
Image acquisition
and
Data analysis

61. Isolate a total RNA containing
mRNA that ideally representsa
quantitative copy of genes
expressed at the time of sample
collection.
 Preparation of cDNA from mRNA
using a reverse- transcriptase
enzyme.
 Short primer is required to initiate
cDNAsynthesis.
 Each cDNA (Sample and Control)
is labelled with fluorescent
cyanine dyes (i.e. Cy3 and Cy5). Fig-03 Sample labeling

62.  Here, the labelled cDNA
(Sample and Control) are
mixed together.
 Purification
 After purification, the
mixed labelled cDNA is
competitively hybridised
against denatured PCR
product or cDNA molecules
spotted on a glass slide.
Fig-04 Array Hybridisation

63.  Slide is dried and scanned to
determine how much
labelled cDNA (probe) is
bound to each target spot.
Hybridized target produces
emissions.
 Microarray software often uses
green spots on the microarray
to represent upregulated genes.
 Red to represent those genes
downregulated
present in
and
equal
that are
yellow to
abundance
Fig-05 Gene chip showing different
type of color spots

64. MICROARRA
YASAGENE
EXPRESSION
PROFILING
TOOL
MICROARRA
Y AS A
COMPARATIV
E GENOMICS
TOOL
DISEASE
DIAGNOSIS
DRUG
DISCOVERY
TOXICOLOGIC
ALRESEARCH

65. The principle aim of using microarray technology as a gene
expression profiling tool is to answer some of the fundamental
questions in biology such as "when, where, and to what
magnitude genes of interest are expressed.
Microarray analysis measure changes in the multigene patterns
of expression to better understand about regulatory
mechanisms and broader bioactivity functions of genes.

66.  Different types of cancer have been classified on the
basis of the organs in which the tumors develop.
 Now, with the evolution of microarray technology, it
will be possible for the researchers to further classify
the types of cancer on the basis of the patterns of gene
activity in the tumor cells.

67.  Microarray technology has extensive application in
Pharmacogenomics.
 Comparative analysis of the genes from a diseased and
a normal cell will help the identification of the
biochemical constitution of the proteins synthesized by
the diseased genes.

68.  Microarray technology provides a robust platform for the
research of the impact of toxins on the cells and their passing
on to the progeny.
 Toxicogenomics establishes correlation between responses to
toxicants and the changes in the genetic profiles of the cells
exposed to such toxicants.
 The microarray permits researchers to examine thousands of
different genes in the same experiment and thus to obtain a
good understanding of the relative levels of expression
between different genes in an organism.

69.  Microarray is a recently developed functional genomics
technology that has powerful applications in a wide
array of biological medical sciences, agriculture,
biotechnology and environmental studies. Since many
universities research institutions and industries have
established microarray based core facilities and
services, microarrays have become a readily accessible,
widely used technology for investigating biological
systems.