power point presentation of Clinical evaluation of strabismus
Polymerase chain reaction
2. 1971- K. Kleppe, E. Ohtsuka, R. Kleppe, I. Molineux∥, H.G.
Khorana, ‘Studies on polynucleotides: XCVI. Repair replication
of short synthetic DNA's as catalyzed by DNA polymerases’.
1984- mullis presented the concept of PCR at cetus corporation.
1985- nature publications and Science rejected mullis paper for
publication.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich
HA, Arnheim N, ‘Enzymatic amplification of beta-globin genomic
sequences and restriction site analysis for diagnosis of sickle
cell anemia’. Science. 1985 Dec 20;230(4732):1350-4. US
Patent 4,683,202.
Mullis, K. B. & Faloona F. A. Specific synthesis of DNA in
vitro via a polymerase-catalyzed chain reaction. Meth.
Enzymol. 155, 335–350 (1987)
3. Mr. Cycle:- first semi automated PCR device
from PerkinElmer Cetus Instruments (PECI)
(1985) collaboration.
First polymerase from E.coli:- Manual
addition of fresh enzyme after every cycle of
denaturation.
Used tubes to heat and cool water
5. Gelfand of cetus isolated taq polymerase
from
thermus acqaticus.
Peltier heating and cooling devices were
used- no manual addition of reagents
(Thermostable Poly).
1989- Patent received by cetus for Taq
polymerase.
8. 1989- agreement of cetus with roche
roche diagnostic systems opened roche diagnostic research
with cetus.
1989-91- 20 licences were made by cetus with commercial
reference labs to perform PCR based diagnostics.
1990- HLA DQ alpha forensic kit:- only product cetus handled
full rights.
1991- roche and perkin elmer announced that roche holds the
ownership of PCR, paying 300 million dollars to cetus.
Peci dissolved and perkin elmer develops instruments and
distributes roche pcr reagents.
Roche opened roche molecular systems (RMS). ROCHE and
perkin elmer made more than 400 licences with other firms to
manufacture PCR reagents and instuments.
9. DNA template
DNA Polymerase
Buffer
Mg++ ions
Primers
Nucleotides
Water
10. The whole genomic DNA, plasmid DNA of bacteria
or viral DNA/RNA ( human genome-3,200 Mb).
Our sequence/gene of interest resides.
Optimal concentration- 0.1-1ug/ml.
Theoretically it is possible to get several copies of
amplicons from a single molecule of DNA.
The result of PCR depends on the quality of
template DNA.
- polymerase inhibitors, proteases, DNAses, etc.
The choice of extraction decides the purity of the
starting material.
11. DNA quantification:-
UV spectrophotometry;
-Principle- beer lamberts law
DNA exibits maximum absorbance of UV at 260 nm.
At 260nm, an absorbance of 1 corresponds to 50ug/ml of DNA.
Purity assesment; the ratio of 260/280 was used. A value of ~1.8
for DNA and ~2 for rna is of PCR grade.
Nanodrop;
- Technical advancement of UV spectrophotometry
- Flourscent based analysers also avail
- Autoanalyse the quality & quantity of DNA. Requires samples in
microvolumes.
Storage; -20 C. frequent thawing and freezing to be avoided to
prevent degradation.
13. Concentration; 0.5 to 3U
Higher concentrations required if the
template contains inhibitors.
Taq- Thermus aquaticus
Pfu-Pyrococcus furiosus ( 3'->5'
exonuclease activity)
recombinant DNA polymerase; eg; platinum
Pfu- high fiedility
14. Provides suitable environment for the
enzyme activity.
Contains,
- mgcl2
-kcl – k+ stabilizes primer annealing
-tris-hcl
Concentration depends on the amount of
mgcl2 present.
Usually 10% i.e 5ul per 50ul reaction
15. Co- factor for polymerase
Act as catalyst
Facilitates the formation of phosphodiester
bonds between nucleotides
Stabilize primer annealing by shielding the
negative electrostatic repulsion between the
primer and template.
Optimum concentration- 1-4mM
Reaction buffers are supplemented along with
mgcl2
16. dATP
dTTP
dCTP
dGTP
Optimum concentration- 0.2mM
Decreased concentration will result in low
quantity of the end product
Increasing the concentration does not result in
increased quantity of finalproduct
17. Allele-specific PCR: a diagnostic or cloning technique based
on single-nucleotide variations (SNVs) (single-base differences
in a patient). It requires prior knowledge of a DNA sequence,
including differences between alleles, and uses primers whose
3' ends containsthe SNV (base pair buffer around SNV usually
incorporated
Assembly PCR or Polymerase Cycling Assembly (PCA):
PCR on a pool of long oligonucleotides with short overlapping
segments. overlapping segments determine the order of the
PCR fragments, thereby selectively producing the final long
DNA product. Oder of genes.
Asymmetric PCR: preferentially amplifies one DNA strand in a
double-stranded DNA template. It is used in sequencing and
hybridization probing where amplification of only one of the two
complementary strands is required
18. Digital PCR (dPCR): measure the quantity
of a target DNA sequence in a DNA sample.
The DNA sample is highly diluted.some of
them do not receive a single molecule of the
target DNA. The target DNA concentration is
calculated.
Helicase-dependent amplification: uses a
constant temperature. DNA helicase, an
enzyme that unwinds DNA, is used in place
of thermal denaturation.
19. Hot start PCR: a reduces non-specific
amplification. heating the reaction
components to the denaturation temperature
(e.g., 95 °C) before adding the polymerase.
Intersequence-specific PCR (ISSR): a
PCR method for DNA fingerprinting that
amplifies regions between simple sequence
repeats to produce a unique fingerprint of
amplified fragment lengths.
20. Inverse PCR: identify the flanking
sequences around genomic inserts. series
of DNA digestions and self ligation, resulting
in known sequences at either end of the
unknown sequence.
Ligation-mediated PCR: uses small DNA
linkers ligated to the DNA of interest and
multiple primers annealing to the DNA
linkers.
23. Miniprimer PCR: uses a thermostable
polymerase (S-Tbr) that can extend from short
primers ("smalligos") as short as 9 or 10
nucleotides. This method permits PCR targeting
to smaller primer binding regions, and is used to
amplify conserved DNA sequences, such as the
16S (or eukaryotic 18S) rRNA gene.
Multiplex-PCR: consists of multiple primer sets
within a single PCR mixture to
produce amplicons of varying sizes that are
specific to different DNA sequences.
24. Nested PCR: increases the specificity of DNA
amplification, by reducing background due to non-specific
amplification of DNA.
Two sets of primers are used in two successive PCRs. In
the first reaction, one pair of primers is used to generate
DNA products, which besides the intended target, may still
consist of non-specifically amplified DNA fragments. The
product(s) are then used in a second PCR with a set of
primers whose binding sites are completely or partially
different from and located 3' of each of the primers used in
the first reaction.
Nested PCR is often more successful in specifically
amplifying long DNA fragments than conventional PCR,
but it requires more detailed knowledge of the target
sequences.
26. Reverse Transcription PCR (RT-PCR): for
amplifying DNA from RNA. Reverse
transcriptase reverse transcribes RNA into cDNA,
which is then amplified by PCR.
RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify
the sequence of an RNA transcript, including
transcription start and termination sites.
If the genomic DNA sequence of a gene is known,
RT-PCR can be used to map the location
of exons and introns in the gene.
28. Based on the detection and quantitation of a
fluorescent reporter
In stead of measuring the endpoint we focus on
the first significant increase in the amount of
PCR product.
The time of the increase correlates inversely to
the initial amount of DNA template
29. Emits a strong fluorescent signal upon binding to double-stranded
DNA
Nonspecific binding is a disadvantage
Longer amplicons create a stronger signal
Denaturation Annealing End of Elongation
31. Amplification can be monitored real-time
No post-PCR processing of products
High throughput, low contamination risk
Requirement of 1000-fold less RNA than
conventional assays
Most specific, sensitive and reproducible
32. 1.Classification of organism based on genetic
relatedness (genotyping)
- Amplification
- Sequencing
- Phylogenetic analysis; multiple sequence
alignment & phylogenetic tree construction.
- MEGA6
34. 2. Identification and confirmation of isolate
obtained from culture
- Amplification of (eg.16s rDNA) sequence
- Sequencing
- BLAST HIT to confirm the organism
- If not avail, to be registered as a new strain
3. Early detection of pathogens in clinical
specimen
- Before the production of antibodies
- Able to detect Very low quantity
35. 4. Rapid detection of antibiotic resistance
- Helps to administer the right antibiotic
5. Detection of mutations
- Mutations would result in the respective alteration
of biochemical activities of microbes.
- Mutation analysis will help to study the
characteristics of genes.
6. Differentiation of toxigenic from non-toxigenic
strains
- By using primers to genes that are responsible for
production of a particular toxin.m
36. The rRNA is the most conserved (least variable) gene in all
cells.
Portions of the rDNA sequence from distantly-related organisms
are remarkably similiar. This means that sequences from
distantly related organisms can be precisely aligned, making the
true differences easy to measure.
Thus the comparison of 16s rDNA sequence can show
evolutionary relatedness among microorganisms.
The 16s rDNA sequence has hypervariable regions, where
sequences have diverged over evolutionary time. These are
often flanked by strongly-conserved regions.
Primers are designed to bind to conserved regions and amplify
variable regions.
38. Analysis and characterization of genes
abnormalities leading to disease.
Understanding genetic diseases pathogenesis
Detection of gene mutation (mutational analysis)
Study of genetic diseases pattern of inheritance
Diagnosis and screening of genetic diseases
Prenatal diagnosis
Identification of diseases carrier to help in genetic
and pre-marriage counseling
39. Many genetic diseases are caused by subtle
changes in individual genes that cannot be
detected by karyotyping.
Traditionally the diagnosis of single-gene
disorders has depended on the identification of
abnormal gene products (e.g., mutant
hemoglobin or enzymes) or their clinical effects,
such as anemia or mental retardation
Now it is possible to identify mutations at the
level of DNA and offer gene diagnosis for
several mendelian disorders.
40. Quantification of gene expression:
Discovery of a new target site for therapeutic
intervention .
Most of the disease pathologies are associated
with an altered gene expression leading to an
increase or decrease in the activity of cellular
proteins.
PCR can be employed to detect very small
alterations in cellular mRNA encoding for such
proteins.
RTPCR
41. Identification purposes
Identify crime suspects
Exonerate persons wrongly accused of crime
Identify crime and catastrophe victims
Establish paternity and other family
relationships
42. RFLP
PCR-RFLP
HLA-DQ
STR analysis
Mitochondrial DNA (mtDNA)
SNP genotyping
VNTRs (variable number of tandem repeats)
43. To measure the compatibility of the donor and recipient in
transplantation.
The PCR test is a new DNA-
based test that can detect the presence
or absence of antigens by determining whether cells have
the genes for the antigens.
An HLA allele is defined by its entire DNA sequence. There
is much sharing of certain polymorphic sequences
between different alleles.
Rapid HLA typing is currently best achieved using
allele‐specific PCR, whereby DNA primers are used to
discriminate between selected sequences in different
alleles.
45. The gold standard test for cancer diagnosis of
almost all tumors is tissue diagnosis.
PCR and/or Southern blot can be used in
diagnosing B and T cell lymphomas.
PCR-based detection of T-cell receptor or
immunoglobulin genes rearrangement allow
distinction between monoclonal (neoplastic) and
polyclonal (reactive) proliferations.
46. Oncogenes; as proto-oncogenes, normally
promote cell division or cell survival.
Oncogene mutations are usually a gain of
function and dominant.
Tumor suppressors: genes normally arrest
cell division.
Tumor suppressor gene mutations are
usually a loss of function and recessive
47. The EGFR oncogene encodes another of the same family
of epidermal growth factor receptors.
This gene is mutated or amplified in several types of
cancer cells.
EGFR gene mutations are detected by SSCP, SSP-PCR,
or direct sequencing.
The 53-kilodalton tumor suppressor gene encodes a
transcription factor.
TP53 is mutated in half of all types of cancer.
Loss of TP53 function is an indicator of poor prognosis in
colon, lung, breast, and other cancers.
TP53 gene mutations are detected by direct sequencing.
1. The concept of single std dna for polymerization . Khorana Explained the steps of repair mechanism similar to PCR. demonstrated the power of PCR as a diagnostic tool before the process has been clearly illustrated by mullis. 1985- Patent applied, 1987- patent receiveed on the name pf mullis for PCR technique.
After the idea of mullis, cetus engineered mr.cycle. To reduce the longevity of the procedure which involves changing tubes between waterbaths. Denaturation inactivates e.coli poly. Mullis work was further standardized at cetus by his colleagues at cetus. He left cetus on 1986.
Mr. cycle- only 3 made- 1- museum of american history, 2. science museum- london, 3. mol.bio dept. univ of southern california.
Roche funds cetus R&D. ROCHE has had the capability to commercialize diagnostic kits world wide.
Sosoc- 2 unit device with base unit and controller. Baby blue- base unit and controller merged
Roche got the right to develop and distribute pcr based diagnostic products by paying royalty to cetus
Inhibitors
K+,NH+,
MICRO, PHARMA, FORENSIC, CANCER, MOLECULAR MEDICINE, PERSONALISED MEDICINE