Transcript DNA - Medical Genetics

Watson and Crick
 The structure of DNA was described by British
Scientists Watson and Crick as long double helix
shaped with its sugar phosphate backbone on the
outside and its bases on inside; the two strand of
helix run in opposite direction and are antiparallel to each other. The DNA double helix is
stabilized by hydrogen bonds between the bases.
Watson and Crick discovers DNA
Feb 28th 1953
DNA
A purine always links with a
pyrimidine base to
maintain the structure of
DNA.
Adenine ( A ) binds to Thymine
( T ), with two hydrogen
bonds between them.
Guanine ( G ) binds to Cytosine
( C ), with three hydrogen
bonds between them.
Nucleic acid probe
 Nucleic acid fragment, labelled by a radioisotope,
biotin, etc., that is complementary to a sequence in
another nucleic acid (fragment) and that will, by
hydrogen binding to the latter, locate or identify it
and be detected; a diagnostic technique based
on the fact that every species of microbe possesses
some unique nucleic acid sequences which
differentiate it from all others, and can be used as
identifying markers or "fingerprints."
Nucleic acid probe
 Nucleic acid fragment, labelled by a radioisotope,
biotin, etc., that is complementary to a sequence in
another nucleic acid (fragment) and that will, by
hydrogen binding to the latter, locate or identify it
and be detected; a diagnostic technique based
on the fact that every species of microbe possesses
some unique nucleic acid sequences which
differentiate it from all others, and can be used as
identifying markers or "fingerprints."
Hybridization probe
 Hybridization probe is a fragment of DNA or
RNA of variable length (usually 100-1000 bases
long), which is used to detect in DNA or RNA
samples the presence of nucleotide sequences (the
DNA target) that are complementary to the
sequence in the probe. The probe thereby
hybridizes to single-stranded nucleic acid (DNA or
RNA) whose base sequence allows probe-target
base pairing due to complementarily between the
probe and target.
Dr.T.V.Rao MD
7
Nucleic Acid Probes
Accu probes from Gene
probe, which contains a
chemiluminescent label
and target the rRNA of
the microorganisms of
interest.
 It reads events in vivo or
during the
multiplication of
organism.
DNA is Endless structure
 The rungs of the ladder
can occur in any order
(as long as the base-pair
rule is followed)
 Those 4 bases have
endless combinations
just like the letters of the
alphabet can combine to
make different words.
DNA Replication
 DNA replication is semi-
conservative. That
means that when it
makes a copy, one half of
the old strand is always
kept in the new strand.
This helps reduce the
number of copy errors.
 So we remained
what we were ?
So we remained what we were
Because of Our Genetic Materials
DNA to RNA
 DNA remains in the
nucleus, but in order for
it to get its instructions
translated into proteins,
it must send its message
to the ribosome's, where
proteins are made. The
chemical used to carry
this message is
Messenger RNA
TYPES OF BLOTTING TECHNIQUES
Blotting technique
Southern Blot
Northern Blot
Western blot
It is used to detect DNA.
It is used to detect RNA.
It is used to detect protein.
Nucleic Acid Hybridizations
 The hybridization of a radioactive probe to
filter bound DNA or RNA is one of the most
informative experiments that is performed in
molecular genetics. Two basic types of
hybridizations are possible.
 Southern hybridization - hybridization of a
probe to filter bound DNA; the DNA is typically
transferred to the filter from a gel
 Northern hybridization - hybridization of a
probe to filter bound RNA; the RNA is typically
transferred to the filter from a gel
Western blotting
 Western blotting is an Immunoblotting technique
which rely on the specificity of binding between a
molecule of interest and a probe to allow
detection of the molecule of interest in a mixture
of many other similar molecules.
 In Western blotting, the molecule of interest is a
protein and the probe is typically an antibody
raised against that particular protein.
 The SDS PAGE technique is a prerequisite for
Western blotting .
Western Blotting
Restriction fragment length
polymorphism
 Sir Alec Jeffreys developed restriction fragment length
polymorphism (RFLP), which quickly became the
standard technique for DNA testing throughout the
1980s. RFLP provided the world with the first form of
genetic testing based on DNA, the body's genetic
material
Restriction Fragment Length Polymorphism
(RFLP)
 Restriction Fragment
Length Polymorphism
(RFLP) is a technique in
which organisms may be
differentiated by analysis
of patterns derived from
cleavage of their DNA.
RFLP
 The resulting DNA
fragments are then
separated by length
through a process known
as agarose gel
electrophoresis, and
transferred to a
membrane via the
Southern blot procedure.
Spoligotyping
 Spoligotyping, a new method for
simultaneous detection and typing of
M. tuberculosis complex bacteria, has
been recently developed. This method
is based on polymerase chain reaction
(PCR) amplification of a highly
polymorphic direct repeat locus in the
M. tuberculosis genome.
Spoligotyping in Tuberculosis
 The well-conserved 36-
bp direct repeats are
interspersed with unique
spacer sequences varying
from 35 to 41 bp in size.
Clinical isolates of MTC
bacteria can be
differentiated by the
presence or absence of
one or more spacers.
Results analyzed by Computer
DNA finger printing
 Every one of our DNA is equal except for only about
0.10 %.
 DNA finger printing lies in uniqueness of those
regions of DNA that do differ from person to person.
 Only 5 % of our DNA code rest do not code called in
past as Junk DNA and contain repeated sequences of
base pairs
 Called as Variable number of
tandem
repeats contain 20 to 100 base pairs and the same
sequence is repeated one to 3 times in a row
Documentation of Finger printing
for Records
 Finger print means translating all the variable number
of tandem repeats to visible records
 All VNTR is tested for restriction length
polymorphism which differ from species to species.
 All the obtained material is blotted to Nylon or
Nitrocellulose membrane ( Southern Blotting )
RLFP to PCR
 Isolation of sufficient DNA for RFLP analysis is time-
consuming and labour intensive. However, PCR can be
used to amplify very small amounts of DNA, usually in
2-3 hours, to the levels required for RFLP analysis.
Therefore, more samples can be analysed in a shorter
time.
Molecular diagnostics – how it works
 Every organism
contains some
unique,
species specific
DNA sequences
 Molecular diagnostics
makes the species
specific DNA visible
PCR methods are rapid and sensitive
 PCR, as a specific,
sensitive and rapid
technique in the
identification of the
pathogen in the clinical
specimen has been
developed extensively
over the past decade. Its
value as a clinical tool is
being increasingly
recognized
Polymerase Chain Reaction Methodology A
Mile stone in Medical History
 He had the idea to use a
pair of primers to
bracket the desired DNA
sequence and to copy it
using DNA polymerase,
a technique which would
allow a small strand of
DNA to be copied
almost an infinite
number of times.
Dr. Kary Mullis, wins Nobel Prize in 1993
 Kary received a Nobel Prize
in chemistry in 1993, for
his invention of the
polymerase chain reaction
(PCR). The process, which
Kary Mullis conceptualized
in 1983, is hailed as one of
the monumental scientific
techniques of the
twentieth century.
PCR Liberates a Innocent Prisoner
 KirkBloods worth
case
 A Waterman
 Imprisoned for 9
years on wrong
evidences of Rape
 Unmatched DNA by
PCR makes a
freeman
Locates Genes for Color Blindness
 Color Blind British John
Dalton died in 1844
 Request his eyes to be
preserved
 And to be investigated why
he confused scarlet with
green, and pink with blue
 Recent PCR studies prove
Dalton lacked a gene for
making one of the three
photo pigments essential for
normal color vision.
Color Blindness is x linked
 The genes for our red
and green colour
receptors are located on
the X-chromosome,
giving women a
redundant set of
receptor genes.
 This is why men are far
more prone to colourblindness than women.
DNA – RNA - DNA
 In Molecular biology, the
polymerase chain
reaction (PCR) is a
technique to amplify a
single or few copies of a
piece of DNA across
several orders of
magnitude, generating
millions or more copies
of a particular DNA
sequence.
Common Tools of Molecular Biology









Nucleic acid fractionation
Polymerase chain reaction
Probes, Hybridization
Vector, Molecular cloning
Nucleic acid enzymes
Microarray
DNA sequencing
Electrophoretic separation of nucleic acid
Detection of genes:
*DNA: Southern blotting; inSitu hybridization; FISH
Technique
*RNA: Northern blotting
*Protein: Western blotting, immunohistochemistry
Current Uses of molecular Biology
The most recent applied technologies, genetic
engineering, DNA finger-printing in the social
and forensic science, pre and postnatal diagnosis
of inherited disease, gene therapy and drug
Design.
Molecular biology allows the laboratory to be
predictive in nature, it gives information that the
patients may be at risk for disease (future).
Major tool in Diagnosis of Infectious
Restriction Endonulease
 If two organisms differ in the distance
between sites of cleavage of a particular
restriction endonuclease, the length of the
fragments produced will differ when the
DNA is digested with a restriction enzyme.
The similarity of the patterns generated
can be used to differentiate species (and
even strains) from one another.
Restriction Endonulease
 Restriction endonucleases are enzymes that cleave
DNA molecules at specific nucleotide sequences
depending on the particular enzyme used. Enzyme
recognition sites are usually 4 to 6 base pairs in length.
 The recognition sequences are randomly distributed
through the DNA and recognizes different nucleotide
sequences, and snips through DNA molecule.
Taq polymerase
 Taq polymerase is a thermos table DNA
polymerase named after the thermophilic
bacterium Thermus aquaticus from which it
was originally isolated by Thomas D. Brock
in 1965. It is often abbreviated to "Taq Pol"
(or simply "Taq"), and is frequently used in
polymerase chain reaction (PCR), methods
for greatly amplifying short segments of
DNA
Disadvantages of Taq Pol
 Taq mis-incorporates 1
base in 104.
 A 400 bp target will
contain an error in 33%
of molecules after 20
cycles.
 Error distribution will be
random.
Thermo cycler is Back bone of PCR
methodology
 The method relies on
thermal cycling,
consisting of cycles of
repeated heating and
cooling of the
reaction for DNA
melting and
enzymatic replication
of the DNA
PCR - Three basic Steps
Cut
Paste
Amplify
PCR Primers
TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT . . . . . . . . . .>
and
<. . . . . . . . . . AAATTTGGCCAA
TTAACGGCCTTAA . . . TTTAAACCGGTT
Cutting, pasting and amplifying is the basis
of Reaction
Denaturing Template
Heat causes DNA strands to separate
5’
3’
3’
5’
Denature DNA strands 94oC
5’
3’
3’
5’
Annealing Primers
•Primers bind to the template sequence
•Taq Polymerase recognizes double-stranded substrate
5’
3’
3’
5’
Primers anneal 64oC
5’
3’
3’
5’
3’
3’
5’
5’
Taq Polymerase Extends
•Taq Polymerase extends primer
•DNA is replicated
3’
5’
3’
5’
3’
3’
5’
5’
Extend 72oC
5’
3’
3’
5’
3’
3’
5’
5’
Repeat denaturing, annealing, and extending 30 cycles
Molecular diagnostics is a set of methods
to study primary structure (sequence) of DNA
•Hybridization with complementary sequences
-A-A-T-T-C-G-C-G-A-T-G- T-T-A-A-G-C-G-C-T-A-C-
•Amplification (synthesis) of species specific sequences
PCR – polymerase chain reaction
-A-A-T-T-C-G-C-G-A-T-G-A-A-T-T-C-G-C-G-A-T-G-A-A-T-T-C-G-C-G-A-T-G-A-A-T-T-C-G-C-G-A-T-G-A-A-T-T-C-G-C-G-A-T-GThe 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
PCR occurs in cycles and Multiplies the DNA
Applications of PCR
The standard
specimen
procedure can
quantitate HIV1 RNA in a range
of 400-75,000
copies/mL.
Advantages of PCR
Speed
Ease of use
Sensitivity
Candida infections can be specifically
identified
The fragments
of 125-bp (EO3)
and 317 bp
(HSP) specific
for C. albicans
were used for
amplification.
Molecular methods proving highly Sensitive
 It has been postulated
that DNA sequencing
of the universal nested
PCR product may allow
identification of the
causative organisms in
a number of culture are
few
PCR helps in several critical Conditions
 PCR has also been evaluated
in the diagnosis of fungal
endophthalmitis using broad
range primers as well as
primers specific for C.
albicans. Detection of fungal
DNA by PCR in intraocular
specimens will prove as a
useful means of diagnosing
endophthalmitis. It will
greatly facilitate management
decisions when conventional
culture is negative.
Advantages
Molecular methods
•High sensitivity and specificity
•Detects pathogen, not immune response
•Quick results
•High transport toleration
In-house (home-brew) PCR methods
•Cost effective
•High sensitivity
•High quality
•Fast implementation of scientific discoveries
•Customer friendly
The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004
QIAGEN One Step RT-PCR Kit
 The QIAGEN One Step
RT-PCR Kit is designed
for easy and sensitive
one-step RT-PCR using
any RNA template. A
unique enzyme
combination and
specially developed
reaction buffer ensure
efficient reverse
transcription and PCR in
one tube.
RT-PCR in one step
The Robus™ T I Kit is base
 RobusT RT-PCR Kits
perform cDNA
synthesis and PCR
amplification of
cDNA successively in
a single tube during a
continuous thermal
cycling
Nested polymerase chain reaction
 Nested polymerase chain reaction is a
modification of polymerase chain reaction
intended to reduce the contamination in products
due to the amplification of unexpected primer
binding sites.
 Nested polymerase chain reaction involves two
sets of primers, used in two successive runs of
polymerase chain reaction, the second set
intended to amplify a secondary target within the
first run products
Loop Mediated Isothermal Amplification
(LAMP)
 Loop mediated isothermal amplification is
a simple, rapid, specific and cost effective
nucleic acid amplification method
characterized by use of 8 distinct regions
on the target gene.
 The amplification proceeds at a constant
temperature using strand displacement
reaction.
Multiplex PCR
 TaqMan probes and
Molecular beacons
allow multiple DNA
species to be measured
in the same sample (
Multiplex PCR) since
fluorescent dyes with
different emission
spectra may be
attached to different
probes
Multiplex PCR in Real Time
 Multiplex real time
quantitative RT-PCR
assays have been
developed for
simultaneous
detection
identification and
quantification of HBV,
HCV and HIV!
 In plasma and Serum
samples.
Prevention of Contamination in PCR
Laboratory
 PCR contamination be considered as a
form of infection. If standard sterile
techniques that would be applied to
tissue culture or microbiological
manipulations are applied to PCR, then
the risk of contamination will be
greatly reduced. Above all else,
common sense should prevail.
Polymerase Chain Reaction Available for
several infections ….
 Chlamydia trachomatis
 Slow growing Mycobacterium
tuberculosis
 viruses like Herpes simplex virus ,
 Varicella Zoster virus .
 Adeno virus in our laboratory for
corneal specimens
Uses and Advantages in Testing by PCR
Methods
 Clinical diagnostics: detection and quantification
of infectious microorganisms, cancer cells and
genetic disorders
 Capable of amplifying long targets, up to 6.0 kb
 One-tube system allows rapid, sensitive and
reproducible analysis of RNA with minimal risk of
sample contamination
 Amplifies products from a wide variety of total
RNA or mRNA sources
Disadvantages of PCR Methods
 Expensive to the Developing world
 Need well trained, Manpower
 Coordination for quality control
 Adoption to changing needs
 Timely technical support
 False positive results due to Amplifications

Over 1400 molecular pathology labs in the US, with bulk of genetic testing being provided by a few big labs.

Large Labs:




Medium Labs:





Quest Diagnostics
LabCorp
Genzyme
Mayo
ARUP (Associated Regional and University Laboratories Pathologist)
MD Anderson
Regional clinics and hospital Laboratories
Key End User product feature requirements:



Clinical Practicality of test
Strong client portfolio and relationship with laboratories
Result turnaround time

Important issue in genetic testing market is deciding which testing platform or technology will have
highest adoption rates in Clinical labs




Technology issues are throughput, sample concentration, procedure sensitivity and specificity
Economic issues are costs of instrument and reagents, current platforms available in the lab, and ease of use of the
procedures
Genetic testing looks for genetic marker such as Single Nucleotide Polymorphism (SNP) , Restriction
Fragment length Polymorphism (RFLP) or short tandem repeat (STR)
Theoretically any marker can be identified through any of the platforms




Real- Time Polymerase Chain Reaction ( RTPC)
Capillary Electrophoresis
Micro-array
Multiplexing Instruments

Important issue in genetic testing market is deciding which testing platform or technology will have
highest rates in Clinical labs






Technology issues are throughput, sample concentration, procedure sensitivity and specificity
Economic issues are costs of instrument and reagents, current platforms available in the lab, and ease of use of the
procedures
Genetic testing looks for genetic marker such as Single Nucleotide Polymorphism (SNP)
Restriction Fragment length Polymorphism (RFLP), or
Short tandem repeat (STR)
Theoretically any marker can be identified through any of the platforms




Real- Time Polymerase Chain Reaction ( RTPC)
Capillary Electrophoresis
Micro-array
Multiplexing Instruments
 Electrophoresis Platform:
 A method of detection for specific gene sequences using gel
electrophoresis
 Currently high availability of electrophoresis instrumentation
in labs means that kits designed for this platform should have
the most rapid uptake.
 Least expensive- $15K US for state of the art equipment
 Electrophoresis also offers low costs per tests - kit cost
$80/patient
 Versatile in number of different application
 Major disadvantage is labor intensity
 RT-PCR Platform:
 A method for rapid and simultaneous amplification,
detection and quantification of gene fragments or gene
expression
 Slower adoption than electrophoresis due to the cost of
machinery - $30K
 Expected to have significant uptake because of
prevalence in SNP testing
 Currently many specialty labs like Quest Diagnostics
have high RT-PCR usage
 Cost expected to drop
 Microarray Platform:
 A method for rapid detection of multiple simultaneous gene
fragments or gene expression. Probes that react to patient’s
genetic material are arranged in grid pattern on glass or
plastic platform. Resulting matches constitute a positive test,
which are readily identifiable.
 Most Expensive $150K-$180K + $500 per test
 Reaction indicate particular genetic sequences, such as those
related to diseases, or how people will respond to certain
medications. Microarrays also can enable researchers to see
which genes are being switched on and off under different
medical conditions.
 Currently does not have many clinical applications, so
drawback for laboratories to invest in platform
 Roche’s AmpliChip is only FDA approved microarray test for
diagnostic use
 Multiplexing Platform:
 A method for simultaneous detection of specific gene
fragments, gene expression, immune response proteins and
enzymes. Multifunction beads are manipulated to bind and
signal the specific presence of a a variety of substrates
 More versatile than microarray
 Allows more variation with regards to type of test - up to 100
assays to be preformed simultaneously on one sample.
 Slower than competing technologies however
 Multiplexing technology are expected to gain field.
 FDA awarded its first approval for a CF diagnostic test to TM
Bioscience’s multiplexing platform based test.
Regulatory Issues



FDA regulates reagents, kits and instruments sold to and performed by clinical labs.
Diagnostic tests can be sold as Analyte Specific Reagents (ASR) to highly sophisticated CLIA-certified
labs. FDA exercised restrain in regulating home- brew tests performed by these labs. No specific
clinical, validity or performance claims are allowed for unregulated ASRs. Potential liabilities if a lab
chooses to provide an ASR rather than a 510(k)-cleared alternative.
Receiving a 510(k) FDA approval is a significant market barrier:





Four FDA approved genetic diagnostic tests:
Roche AmpliChip, used to individualize dosage of antidepressants, antipsychotics, beta-blockers, and
some chemotherapy drugs
TM Bioscience (Toronto, Canada) TAG-IT assay for detecting cystic fibrosis
Visual Genetics (Toronto, Canada acquired by Bayer in ‘02) TRUGENE HIV-1 Genotyping Kit, used to
detect variations in the genome of the human immunodeficiency virus that make the virus resistant
to some anti-retroviral drugs.
Third Wave’s Invader assay detects variations in a gene that produces the enzyme UDPglucuronosyltransferase, and predicts adverse events.
 http://www.fda.gov/cdrh/oivd/index.html
http://www.fda.gov/cder/genomics/default.htm