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Molecular diagnostic
technologies-1
Manipulation of nucleic acids
and amplification of genetic
material
ZAHRA HASAN, PhD
Associate Professor
Section of Molecular Pathology 1
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Objectives of this class
• To relate the question (target genetic
material) with the assay required
• To understand how polymerase chain
reaction works
• To understand the methods for visualising
PCR products
• To understand conventional and real time
PCR
• To differentiate qualitative and quantitative
PCR 2
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Amplification
• Why ?
• How ?
• What – material ?
• Which – protocol ?
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Extraction of nucleic acid from
clinical specimen• Viral or bacterial sample
• Human genetic material
• Source of sample
– Blood / serum
– Body fluids:
– pulmonary (BAL, sputum)
– extrapulmonary specimen (ascites, CSF,
Pleural fluid, pus)4
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Manipulation of DNA/RNA• Lysis of cell walls
– DNA/RNA release after cellular lysis using chemical (SDS) or enzymatic (Proteinase K, lysozyme)
• Extraction of DNA/RNA from nucleo-proteins– Use of prepared spin column
– Extraction using a salting out method
• Isolation / Precipitation of nucleic acids by Salting out and centrifugation
• Ethanol precipitation (EtOh increases
hydrophobicity)
• Salts increases ionic strength of solution (NaCl,
NH4Ac, LiCl)5
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• Protection from nucleases
(DNAses – degraded at 95 C; RNAses are not degraded)
Store appropriately (temp, sterile)
-20 C for longer term storage, 4 C for short term (week)
Storage of DNA
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Quantification of nucleic acids
Spectrophotometric measurement
based on Optical Density
Nucleic acids have maximum absorption at 260nm
Proteins have max absorption at 280 nm
Purity determined by DO 260/280 ratio
ODs of pure solution
DNA 50 mg/ml OD 260/280 = 1.8
RNA 40mg/ml OD260/280=2.0
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Nucleic acid based amplification methods
PCR
Conventional – end point analysis
Real-time PCR – kinetic flourescence analysis
Qualitative
Quantitative
Nested- PCR
ARMS PCR
PCR –RFLP
Melting curve analysis for a) differentiation, b) SNP analysis
Ligase Chain Reaction
Reverse line probe assay
Transcription mediated amplification- hybrid capture
Strand displacement assay
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Utility of PCR
• the detection and diagnosis of infectious
diseases
• the diagnosis of hereditary diseases
• For DNA cloning for DNA sequencing
• DNA-based phylogeny, or functional analysis of
genes;
• the identification of genetic fingerprints (used in
forensic sciences and paternity testing); and
• For determining concentration of target (viral
load; mutation load) 9
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DNA replication
Strands complementary and anti-parallel
One strand built 5’ – 3’ direction
Other strand built 3’ – 5’ direction
Each strand can be template for other
Semi-conservative replication
Helicase
DNA polymerase
DNA ligase
primase
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Requirements for PCR
• Primers designed to known sequence ‘flanking’
unknown region, 18-30 bp in size
• DNA polymerase from Thermophilus aquaticus
• dNTPs
• MgCl2
correct Tm
multiple cycles
Automated PCR cycler
CONTROLS, +ve and -ve
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95C
72C
56C
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Careful primer design is key!
• length of primers
– 18-25 bp for PCR assay
• Size of expected product depends on assay (50-150
bp for real time PCR)
• Specific and stringent
• high efficiency
• no primer-dimers
• Choice of polymerase – depends on PCR product
size and also the GC content of the target nucleic
acid sequence
• End of primer should be A or T and fewer GC strings
• Primers should have similar Tm (melting temp) 13
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Tm of primers should be similar
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Factors determining ‘Tm’: the
temperature at which 50% of all
sample is in a denatured form
• Strand length- determines H bonding
• Base composition- more bonds in GC,
>GC% requires higher temp.
• Chemical environment – Na+ stabilises
helix while urea and formamide destabilise
it
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Example; selection of primers
for PCR of region of interest
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Design based on the positive strand of DNA
Forward primer is 5 to 3 ‘ direction
Reverse primer is 3’ – 5 ‘ direction
5 ‘ ---------------------3’
3 ‘ ---------------------5’
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RT-PCR
• RNA as starting material – reverse
transcription required
• RNA viruses -CCHF, HIV
• Human Gene expression – oncogenes
• Study of transcriptional responses
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IMPORTANCE OF RNA
QUALITY• Should be free of protein (absorbance
260nm/280nm)
• Should be undegraded (28S/18S ~2:1)
• Should be free of DNA (DNAse treat)
• Should be free of PCR inhibitors
– Purification methods
– Clean-up methods
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Storage of RNA• Store RNA at – 70 C
• RNAses are extremely stable and refold after denaturation
• Inhibit cellular RNAses
• Separate RNA from DNA and proteins
• Keep RNAse out
• RNAses can be degraded by Diethylpyrocarbonate (DEPC) /RNAse away
• Use RNAse inhibitor in reactions
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EXON 1 EXON 2INTRON 2 DNA
EXON 1 EXON 2 RNA
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RT-PCR based transcriptional analysis of genes
- looking at human gene expression; e.g. for oncological changes (Bcr gene)
- primers should be designed appropriately
Ideally should not give a DNA signal cross exon/exon boundary
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OVERVIEW
RT-PCR
tissue
extract RNA
copy into cDNA
(reverse transciptase)
PCR
analyze results21
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Importance of controls
• negative control (no DNA)
– checks reagents for contamination
• no reverse transcriptase control
– detects if signal from contaminating DNA
• positive control
– checks that reagents and primers work
– especially importance if trying to show absence of expression of a gene
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Importance of cleanliness in
PCR• Contamination is major problem
• Huge amplification contributes to this
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Electrophoresis - properties
• Nuc. Acids are charged mols and migrate
in the direction of the electrode with
opposite charge (anode)
• Electrophoretic mobility:
– dsDNA migrates inversely proportional to
log10 of bp
– Base composition, conc. of gel, composition
and ionic strength of buffer, temperature and
intercalating dyes
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DNA separation
Agarose gel electrophoresis:
visualisation with ethidium bromide (UV illumination required)
SYBR green dye (fluorescence measurement)
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Separation on agarose gels depends on
1. Size
2. Electrophoretic mobility
3. The agarose concentration
Smaller molecules travel more rapidly
Larger molecules migrate more slowly
Concentration of agarose is determined by size of product of interest
0.8% gel for products 1 – 10 kb
1% agarose for products 500bp to 2000bp
2% agarose for sizes 50 – 500 kb
NOTE
It is important to use DNA size ladders to run alongside samples on a
Gel in order to size the PCR products/DNA
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How are you going to measure
the PCR product• Directly
– Ethidium bromide staining
– Sybr green dye
• Indirectly
– In addition to primers, add a fluorescently
labeled hybridization probe
– Second set of probes
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Analysis of conventional PCR
based on agarose gel
1 2 3 4 5 6 +C -C
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End point analysis
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REAL TIME PCR
USING SYBR GREEN
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REAL TIME PCR
• kinetic approach
• early stages
• while still linear
www.biorad.com30
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www.biorad.com
2a. excitation
filters
2b. emission
filters
1. halogen
tungsten lamp
4. sample plate
3. intensifier5. ccd
detector
350,000
pixels
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0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBERA
MO
UN
T O
F D
NA
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
33
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SERIES OF 10-FOLD DILUTIONS34
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Importance of controls
• negative control
– checks reagents for contamination
35
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Amplification
Qs. WHAT IS THE VALUE OF USING A PROBE IN THE
REAL-TIME PCR ASSAY?
Ans. The signal is not always specific
to the target sequence
36
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Example; selection of primers
for PCR of region of interest
37
Design based on the positive strand of DNA
Forward primer is 5 to 3 ‘ direction
Reverse primer is 3’ – 5 ‘ direction
5 ‘ ---------------------3’
3 ‘ ---------------------5’
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DNA probes on the principle of
Nucleic acid hybridisation
• Involves mixing of two sources of nucleic
acids, a probe which typically consists of a
homogenous population of identified
molecules (i.e, synthesized oligos) and a
target, which consists of a complex,
heterogenous population of nucleic acid
molecules
38
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Nucleic acid probes detect a Unique Target
Sequence - RNA or DNA
Must be single stranded to hybridise to target
Short- 15-50 nucleotides
Labeled at 5’ or 3’ end:
Radioactive methods - P32, S35
Non-radioactive methods –
biotin-streptavidin, digoxygenin
Fluorescent labels; fluorescein, rhodamine, texasred
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Fluorophore colour Excitation max Emission max.
Fluorescein green 494 523
Rhodamine red 555 580
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Real-time PCR assays-1
TaqMan assay -A TaqMan probe has 5’ Reporter dye and a 3’ dye
Quencher
PRIMERS (F and R) bind
Probe binds upstream to PRIMERS
DNA pol will synthesize new strand,
Extend to Probe site
CLEAVE off the ®
The cleaved ® will flouresce
Fluorescence will be detected
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Real-time PCR assays-2
FRET probe assays – Flourescence Resonance Energy Transfer
between a Donor and an Acceptor probe
PRIMERS (F and R) bind
PCR product generated
Probes bind to internal sequence of
PCR product
Probe 1- 5’ Red label/640nm – with 3’
Phosphate
Probe 2- 3’ Flourescein
Hybridisation- Fluor excited and
transfers energy to
Red Acceptor probe
Red Fluorescence will be detected
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Real-time PCR assay-3
A Molecular beacon has a Donor
And an acceptor in a stem loop
(Closed) conformation
These are good for detection of small sequence changes/single mutations
As they only hybridise when there is a perfect match at the Target
Hybridisation will result in separation between FLOUR and QUENCHER
FLOURescence will now be emitted and detected by machine
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Use of FLOURESCENT PROBES IN REAL TIME PCR
MAKES reaction SPECIFIC!44
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Types of PCR
• Qualitative PCR
– Allows determination of presence or absence
of target gene
• Quantative PCR
– Allows determination of specific quantity of
target as compared with quantification
standards, e.g. viral load determination
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Types of PCR
• Quantitative
For viral load determination, HCV, HSV, HIV,
HDV, HBV
Treatment response – therapy and progress
For extent of mutation – such as Bcr/Abl
mutation in Chronic myeloid leukemia
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15SERIES OF 10-FOLD DILUTIONS
threshold
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Advantages
•sensitive
•quick
•provides a high copy of genes
•a small sequence can be targeted
Disadvantage
false positives
PCR
48
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APPLICATIONS of PCR
•DIAGNOSIS, identification of pathogen
•SPECIATION, e.g. M.avium/ M. intracellulare
•DETECTION OF DRUG RESISTANCE
•IDENTIFICATION OF TOXINS, e.g. Clostridium
•EPIDEMIOLOGICAL STUDIES
genotyping, ribotyping
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Advantages and Disadvantages of
Realtime PCR assays
• Advantage
– Highly specific and sensitive
– Multiple probe labels (530/640 /705nm) allow
multiplexing of signals and detection of target
and control DNA
– Disadvantage – relatively expensive and
specialised equipment required
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Summary of this class
• To relate the question (target genetic
material) with the assay required
• To understand how polymerase chain
reaction works
• To understand the methods for visualising
PCR products
• To understand conventional and real time
PCR
• To differentiate qualitative and quantitative
PCR 51