brief discussion on principle,techniques & applications

1. 1
Presented by
Dr. Tausia Nasreen
PCR,RTPCR,FISH
Principle, Technique & Applications

2. Introduction to molecular
cytogenetics
Polymerase Chain Reaction
RT-PCR
Fluorescent In-situ Hybridization
Applications

3. What Is Cytogenetics……?
Genetics - The branch of science which deals
with the study of heredity and the variation of
inherited characteristics.
Cytology - The branch of science which deals
with the study of structure and function
of cell and cell organelles.
Cytogenetics - The branch of science which deals
with the study of inheritance in relation to the
structure and function of chromosomes.

4. Human have
tendency to
reveal the
secrets behind
every thing…..

5. • The first illustration of human chromosomes
• First used the term mitosis1882
• Waldeyer introduced the term Chromosome
• Greek word for colored body1888
• Sutton combined two disciplines, cytology and
genetics as Cytogenetics1902
• Levitsky termed the ordered arrangement of
chromosomes as karyotype1924
• Caspersson and co-workers reported a unique
banding pattern
• Drets and Shaw produced banding pattern by
Giemsa stain
1971
• Pardue and Gall demonstrated in situ hybridization.
1969
• Tjio and Levan started human genome project1956

6. •Albertson detected biotin labelled probes
1983
• Kary Mullis conceived PCR1983
• Guan used chromosome microdissection for
whole chromosome painting probes (WCPs)1994
• Speicher developed multiplex fluorescence in situ
hybridization (M-FISH)
• Schröck developed spectral karyotyping (SKY)
1996
• Kjell Kleppe et al first described a method using an
enzymatic assay to replicate a short DNA template with
primers in vitro.
1971
•Langer developed non isotope labelling of DNA by
conjugation of biotin1981

7. PCR invented in 1983:
• Kary Mullis at Cetus Corp.
• “Enzymatic application” used to amplify small DNA
fragments
• Diagnostic to genotype Sickle Cell Anemia (b-globin gene)
• In 1993 Kary Mullis won Nobel Prize
Revolutionary technique:
• Amplifies > 1 billion copies of DNA from ONE template
molecule
• One day to genotype patient (mutant or wild-type allele?)
(much faster than Southern blot which takes days!)

9. Polymerase Chain Reaction (PCR)
• PCR is a technique which is used to amplify the number of
copies of a specific region of DNA, in order to produce
enough DNA to be adequately tested.
• The purpose of a PCR is to make a huge number of copies of
a gene. As a result, it now becomes possible to analyze and
characterize DNA fragments found in minute quantities in
places like a drop of blood at a crime scene or a cell from an
extinct dinosaur.
• PCR is valuable to researchers because it allows them to
multiply unique regions of DNA so they can be detected in
large genomes.

10. PCR (Cont’d)
• When first developed, multiple cycles of the PCR
process were cumbersome for two reasons:
• First, the DNA polymerases (Klenow fragment)
available at the time were inactivated each time the
temperature was raised to denature the template strand.
• Second, three water baths at three different temperatures
were necessary, which meant that constant human
attention was required.

11. Two developments were instrumental in the
maturation of the PCR process.
• First was the purification of a heat-stable DNA
polymerase (Taq DNA polymerase).
• The second development was the invention of a
thermal cycler.

14. Technique
• A PCR is performed on an automated cycler, which heats
and cools the tubes with the reaction mixture in a very short
time.
• Performed for 30-40 cycles, in three major steps:
1)Denaturation
2)Annealing
3)Extension

15. •
What is PCR? :
The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence to be
amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength
of the reaction solution suitable for the activity of
the enzyme

16. DNA template
• DNA containing
region to be
sequenced
• Size of target DNA to
be amplified : up to
3 Kb

17. Primers
• 2 sets of primers
• Generally 20-30 nucleotides long
• Synthetically produced
• In order to use PCR, one must know the sequences
which flank both ends of a given region of interest in
DNA. One need not know the DNA sequence in-
between.

18. 5’ 3’
3’ 5’
F
R
•Complementary to opposite
strands with 3’ ends pointing
towards each other
•Not complimentary to each
other
•Should have similar melting
temperatures
•Be in vast excess

19. Deoxyribonucleotides
Nucleotide sequences of the genes are determined
by the precise order of appearance of 4 different
deoxyribonucleotides within a stretch of DNA.
 The four nucleotide bases, the building blocks of
every piece of DNA, are represented by the letters
A, C, G, and T, which stand for their chemical
names: adenine, cytosine, guanine, and thymine.
 The A on one strand always pairs with the T on the
other, whereas C always pairs with G.

20. Equipments required
• PCR machine
• Biosafety cabinet
• 96 well plates
• Pipettes
• Microcentrifuge
tubes
• Table top centrifuge
• Tips with tips boxes
• Tissue paper
• Cool box

22. Enzyme
• Usually Taq Polymerase or
anyone of the natural or
Recombinant
thermostable polymerases
• Stable up to 95◦ C
• High processivity
• Taq Pol has 5’-3’ exo only,
no proofreading

26. • Tm = 4 (G + C) + 2 (A + T) ° C
• Tm of Primer is the melting temperature of the
less stable primer-template pair.
• Tm of Product is the melting temperature of the
PCR product.

32. Standard Thermocycle

33. • At the end of a PCR, the product must be checked before it is used in
further applications. This is to confirm:
• There is a product formed: Not every PCR is successful. There is a
possibility that the quality of the DNA is poor, that one of the primers
doesn't fit, or that there is too much starting template.
• The product is of the right size: It is possible that there is a product, for
example a band of 500 bases, but the expected gene should be 1800 bases
long. In that case, one of the primers probably fits on a part of the gene
closer to the other primer. It is also possible that both primers fit on a
totally different gene.
• Only one band is formed: As in the description above, it is possible that
the primers fit on the desired locations, and also on other locations. In
that case, you can have different bands in one lane on a gel.

35. • The ladder is a mixture of fragments with known size to compare with the
PCR fragments. Notice that the distance between the different fragments of
the ladder is logarithmic. Lane 1 : PCR fragment is approximately 1850
bases long. Lane 2 and 4 : the fragments are approximately 800 bases long.
Lane 3 : no product is formed, so the PCR failed. Lane 5 : multiple bands are
formed because one of the primers fits on different places.

36. Technical steps
1. Keep all reagents in Aliquots of 5-10µl volumes.
2. ALWAYS make a worksheet before starting PCR reaction.
3. Clean the biosafety cabinet with 70% ethanol. AVOID touching
pipettes’ tips as it inhibits amplification.
4. Keep buffer, dNTPs, MgCl2 and water at room temp., thaw, vortex
well.
5. Keep Taq pol. in cool box or -20°C freezer until needed.
6. Label the PCR tubes with sample no.s and keep them in rack.
7. Add requried vol. of water to PCR tube.
8. Prepare MASTER MIX in a separate tube by mixing required vol. of
reagents
• Add 10X PCR buffer, dNTPs, MgCl2, forward and reverse primers.
• Remove Taq enzyme from freezer and add to master mix.
9. Gently centrifuge the master mix and distribute to each PCR tube.
10. Add required vol. of DNA.
11. Centrifuge again to remove bubble.
12. Place PCR tubes in PCR machine.

37. PCR BASED METHODS
• High performance liquid chromatography
• Restriction fragment length polymorphism
• Real-time/ High resolution melt curve analysis
• Amplification – refractory mutation systems
• Digital PCR

38. Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR)
• Reverse transcriptase is a common name for an enzyme that functions as a
RNA-dependent DNA polymerase. They are encoded by retroviruses, where
they copy the viral RNA genome into DNA prior to its integration into host
cells. In the laboratory, it is used for analysing gene expression. i.e. convert
mRNA to cDNA by reverse transcription.
• Reverse transcriptases have two activities:
• DNA polymerase activity
• RNase H activity
• All retroviruses have a reverse transcriptase, but the enzymes that are
available commercially are derived from one of two retroviruses, either by
purification from the virus or expression in E. coli:
• Moloney murine leukemia virus
• Avian myeloblastosis virus

39. The technique consists of two
parts:
1) The synthesis of cDNA
(complementary DNA) from
RNA by reverse transcription
(RT)
2) The amplification of a specific
cDNA by PCR RNA-directed DNA
polymerase (rTh) Yields ds cDNA

41. Applications
• Genome mapping and gene
function determination
• Biodiversity studies ( e.g.
evolution studies)
• Diagnostics ( prenatal testing of
genetic diseases, early detection
of cancer, viral infections)
• Detection of drug resistance
genes
• Forensic (DNA fingerprinting)

42. Advantages
• Automated, fast, reliable
(reproducible) results
• Contained :(less chances of
contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols

43. Disadvantages
• Need for equipment
• Taq polymerase is expensive
• Contamination
• False reactions
• Internal control
• Cross-reaction
• Enrichment steps in (contaminated) samples
• Capacity building needed
• Unspecific amplification

44. Detection Of Pathogens
Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul
et al.1994)
Molecular Identification:

45. Quantitation of mRNA
• Northern blotting
• Ribonuclease protection assay
• In situ hybridization
• cDNA arrays
• PCR
- most sensitive
- technically simple
- can discriminate closely related
mRNAs
- but difficult to get truly
quantitative results

46. Real-time Quantitative PCR
• Same as PCR, but measures
the abundance of DNA as it
is amplified.
• Useful for quantitatively
measuring the levels of
mRNA in a sample.
• Uses reverse transcriptase
to generate cDNA for the
template.

47. • Real-time PCR can be used quantitatively (Quantitative real-time
PCR), semi-quantitatively, i.e. above/below a certain amount of
DNA molecules (Semi quantitative real-time PCR) or qualitatively
(Qualitative real-time PCR).
• Two common methods for the detection of PCR products in real-
time PCR are: (1) non-specific fluorescent dyes that intercalate with
any double-stranded DNA, and (2) sequence-specific DNA
probes consisting of oligonucleotides that are labelled with
a fluorescent reporter which permits detection only
after hybridization of the probe with its complementary sequence.

48. Real-time PCR with double-stranded
DNA-binding dyes as reporters
• A DNA-binding dye binds to all double-stranded
(ds) DNA in PCR, causing fluorescence of the dye.
• An increase in DNA product during PCR therefore leads to
an increase in fluorescence intensity measured at each cycle.
• However, dsDNA dyes such as SYBR Green will bind to all
dsDNA PCR products, including nonspecific PCR products
(such as Primer dimer).
• This can potentially interfere with, or prevent, accurate
monitoring of the intended target sequence.

49. SYBR Green binds
to double-stranded
DNA
and fluoresces

50. This method has the advantage of only needing a
pair of primers to carry out the amplification,
which keeps costs down
However, only one target sequence can be
monitored in a tube.

51. Fluorescent reporter probe
method
• Fluorescent reporter probes detect only the DNA containing the
sequence complementary to the probe.
• Use of the reporter probe significantly increases specificity, and
enables performing the technique even in the presence of other
dsDNA.
• Using different-coloured labels, fluorescent probes can be used in
multiplex assays for monitoring several target sequences in the
same tube.
• The specificity of fluorescent reporter probes also prevents
interference of measurements caused by primer dimers, which are
undesirable potential by-products in PCR.

52. • The method relies on a DNA-based probe with a fluorescent
reporter at one end and a quencher of fluorescence at the
opposite end of the probe.
• The close proximity of the reporter to the quencher prevents
detection of its fluorescence.
• Breakdown of the probe by the 5' to 3' exonuclease activity
of the Taq polymerase breaks the reporter-quencher proximity
and thus allows unquenched emission of fluorescence, which
can be detected after excitation with a laser.
• An increase in the product targeted by the reporter probe at
each PCR cycle therefore causes a proportional increase in
fluorescence due to the breakdown of the probe and release of
the reporter.

56. • During the exponential amplification phase, the quantity of the
target DNA template (amplicon) doubles every cycle.
• However, the efficiency of amplification is often variable among
primers and templates.
• Therefore, the efficiency of a primer-template combination is
assessed in a titration experiment with serial dilutions of DNA
template to create a standard curve of the change in cycle
threshold with each dilution.
• The cycle threshold method makes several assumptions of reaction
mechanism and has a reliance on data from low signal-to-noise
regions of the amplification profile that can introduce substantial
variance during the data analysis

58. Real-time Quantitative PCR
amplification plot

62. The principle of Taqman qPCR

63. What Type of Instruments
are used with Real-Time
PCR?
Real-time PCR systems consist of THREE main
components:
1.Thermal Cycler (PCR machine), linked to a…
2.Optical Module (to detect fluorescence in the tubes
during the run), linked to a…
3.Computer (to translate the fluorescence data into
meaningful results).

64. A good example is the
MiniOpticon real-time
instrument.
Optical Module
Thermal Cycler Base

65. Trouble-Shooting
• A successful real-time PCR experiment will have the
following characteristics:
Replicates are
tightly clustered
Baselines are
relatively flat
Dilution series has
expected spacing
Plateau height
doesn’t matter
Melt curve has one
peak per product.
Curves are all
S-shaped
Curves are
smooth

66. Real-Time
PCR in
Gene
Expression
Analysis
Example: BRCA1 Expression Profiling
BRCA1 is a gene involved in tumor suppression.
BRCA1 controls the expression of other genes.
In order to monitor level of expression of BRCA1, real-
time PCR is used.
DNA
mRNA
Protein
BRCA1
Determine gene
expression

67. Real-Time
PCR in
Disease
Management
Example: HIV Treatment
Drug treatment for HIV infection often depends on
monitoring the “viral load”.
Real-Time PCR allows for direct measurement of the
amount of the virus RNA in the patient.
Viral RNA
Measure amount
of virus, adjust
prescriptions.

68. Fluroscent in situ hybridization
(FISH)

69. In situ hybridization
• A method of localizing and detecting specific mRNA sequences
in morphologically preserved tissues sections or cell
preparations by hybridizing a nucleotide probe to the sequence
of interest.
• The principle: specific annealing of a labelled nucleic acid
probe to complementary sequences in fixed tissue, followed by
visualisation of the location of the probe.
• A critical aspect of these procedures is that the target nucleic
acid is retained in situ
• The sensitivity: 10-20 copies of mRNA per cell.

70. Causes Of Chromosomal
Abnormalities
Chromosomes Number
Chromosomes Size
The Arrangement Of
The Chromosome
Segment
No. Of Chromosome
Segment
Variation in
chromosome
morphology
Variation In

71. Variation In Chromosome Number
• A general term for the variation in the
no.
• Aneuploidy
• Chromosomes no. that are not exact
multiples of n.
• It is further classified as
1. Monosomic:
2. Trisomic:
3. Tetrasomic:
4. Double trisomic:
5. Nullosomic:

73. Euploidy / Polyploidy
• Increase in chromosomes numbers due to 1
or more complete sets of Chromosomes is
called as euploidy.
1. Monoploid:
2. Diploid:
3. Triploid:
4. Tetraploid:
5. Autotetraploid:
6. Polyploid:

75. Variation In No. Of Chromosome
Segment
• Deletion
• It is a common phenomenon of a chromosome to break.
Two types of deletions are possible
1. Terminal
2. Intercalary
• Duplication:
• Large or small piece of chromosome containing extra
blocks of genes due to duplication are found in many
individuals or even in races.

76. Variation in chromosome
morphology
1. Isochromosomes
2. Bridge-breakage –fusion-bridge cycles:
3. Ring chromosome:

77. ABNORMALITIES OF GENES/DNA
1. Point mutation
2. Gross mutation
3. Nonsense mutation
4. Silent mutation
5. Missense mutation
6. Neutral mutation
7. Frameshift mutation
8. Structural mutation

79. PREVENTION IS BETTER THAN
CURE….
!!

80. What is FISH…..?
• Fluorescence in situ hybridization (FISH)
is a cytogenetic technique that
uses fluorescent probes that bind to only
those parts of the chromosome with a
high degree of sequence
complementarity
• It is used to detect and localize the
presence or absence of
specific DNA sequences on chromosomes
.
• FISH is often used for finding specific
features in DNA for use in genetic
counseling, medicine, and species
identification

81. What are probes
• Probe is a synthesized fragment of DNA or RNA of
variable length which can be radioactively labeled
• The size may be varies from 100-1000 bases long.
• The probe thereby hybridizes to single-stranded nucleic
acid (DNA or RNA) whose base sequence allows probe-
target base pairing due to complementarity between the
probe and target.
• The probe is tagged (or "labeled") with a molecular
marker of either radioactive or fluorescent molecules.

82. Fluroscent In Situ Hybridization
• Step I – Denaturation
Conversion of double stranded dna in to single stranded
dna
• Step II – Hybridization
Application of probe DNA to slide & overnight incubation
at 37°C
Binding of probe DNA to trarget DNA.
• Step III – Post hybridisation washing & detection
Washing of unbound probe DNA.
Application of counter stain &
• Step IV – counter stain
Application of counter stain.
• Step V – Visualization
visualization using fluorescence microscopy.

84. Slide With Metaphase
Chromosomes
Denaturation Of DNA
Addition Of Probe
Hybridization
Washing And
Addition Of Counter
Stain
Visualization

86. Choice of probe
Probes are complimentary sequences of nucleotide bases to the
specific mRNA sequence of interest. These probes can be as
small as 20-40 base pairs or be up to 1000 bp.
The strength of the bonds between the probe and the target
decreases in the order RNA-RNA to DNA-RNA. This
stability is influenced by the various hybridization conditions
(salt concentration, hybridization temperature, concentration
of formamide, pH).

87. Probe types
• Oligonucleotide probes
• Single stranded DNA probes
• Double stranded DNA probes
• RNA probes (cRNA probes or
riboprobes)

88. Oligonucleotide probes
Produced synthetically by an automated chemical synthesis.
Advantages:
• Small (40-50 base pairs; easy penetration into the cells or
tissue of interest).
• Resistant to RNases
• Single stranded (no renaturation).

89. Single stranded DNA probes
• Similar advantages to the oligonucleotide probes
• Larger (200-500 bp size range).
• Produced by reverse transcription of RNA or by amplified
primer extension of a PCR fragment in the presence of a single
antisense primer.
• Disadvantages: time to prepare, expensive reagents used, good
repertoire of molecular skills required for their use.

90. Double stranded DNA probes
• The sequence of interest inserted in bacteria, cloned and the
sequence excised with restriction enzymes.
• Because the probe is double stranded, denaturation has to be
carried out prior to hybridization in order for one strand to
hybridize with the mRNA of interest.
• These probes generally less sensitive (DNA strands tend to
rehybridize to each other)
• Not as widely used today

91. RNA probes (cRNA probes or riboprobes)
The most widely used probes with in situ hybridization.
Advantage: RNA-RNA hybrids thermostable and resistant to
digestion by RNases.
Two methods of preparing:
• RNA polymerase-catalyzed transcription of mRNA
• In vitro transcription of linearized plasmid DNA with RNA
polymerase.
Disadvantage: difficult to prepare, sensitive to RNases, poor tissue
penetration

92. Benefits of using oligonucleotide probes
1. Stability
2. Availability
3. Faster and less expensive to use
4. Easier to work with
5. More specific
6. Better tissue penetration
7. Better reproducibility

94. Types of FISH….?
Interphase FISH
Telomeric FISH
RNA in situ
hybridization
Prime in situ
labelling
COBRA FISH
SKY FISH
M- FISH
Fibre FISH

95. Spectral Karyotyping (SKY)

96. How to do SKY….?
Visualization
Image Acquisition
Washes And Detection
Application Of Dye
Hybridization
Probe Denaturation
Denaturation Of DNA
Chromosome Preparation From Tissue
Same as
FISH

97. Spectral Karyotype Observation

98. Application
• FISH generally used compliment classical staining technique
• Substitute for chromosome identification at metaphase or
interphase
• Useful in several clinical settings to determine prognosis and
detection of genetic abnormalities like anupolidy,
characteristic gene fusion, cross of chromosomal region or
whole chromosom.
• Discrete information is obtained from each cell

99. • Helps in detection of single gene disorder and
presence or absence of particular gene on
chromosome.
• By using probe chromosomal material of unknown or
uncertain organism can be identified
• FISH is powerful technique use in detection of
chromosomal abnormalities
• Most significance advance in both research like gene
mapping or identification of noval oncogenes or
genetic abberation and diagnosis of haematological
malignancies and solid tumor.

100. HER2 amplified HER2 non-amplified
Acquired from Vysis Educational Slide Set

101. Her2/neu probes
• LSI- locus specific identifier, specific for her-2 gene
locus
• CEP- chromosome enumeration probe, specific for
α satellite DNA sequence at the centromeric region
of chromosome 17

102. Interpretation of HER-2/neu
• A minimum of 20 invasive tumor cells should be counted.
• Positive -
• Single probe HER2 copy is at least 6 signals/cell.
• Dual probe HER2/CEP17 ratio is at least 2, with HER2 copies being at
least 4 signals/cell OR
• Dual probe HER2/CEP17 ratio is less than 2, with HER2 copies being at
least 6 signals/cell
• Equivocal-
• Single probe HER2 copy is 4- 6 signals/cell.
• Dual probe HER2/CEP17 ratio is less than 2, with HER2 copies being 4-6
signals/cell
• Negative-
• Single probe HER2 copy is <4 signals/cell.
• Dual probe HER2/CEP17 ratio is less than 2, with HER2 copies being <4
signals/cell

103. FISH to Identify NPM/ALK Fusion
Gene
103