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Polymerase chain reaction
 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)
 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
Polymerase chain reaction
 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.
Son-of-son-of-Cycle Baby Blue
First device with Peltier heating
and cooling system
First integrated peltier-based
thermal cycler.
First commercial
PCR device
launched by PECI
 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.
 DNA template
 DNA Polymerase
 Buffer
 Mg++ ions
 Primers
 Nucleotides
 Water
 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.
 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.
Polymerase chain reaction
 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
 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
 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
 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
 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
 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.
 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.
 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.
Polymerase chain reaction
Polymerase chain reaction
 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.
 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.
Polymerase chain reaction
 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.
Polymerase chain reaction
 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
 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
 Taqman probes
 Molecular beacons
Denaturation Annealing Elongation
 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
1.Classification of organism based on genetic
relatedness (genotyping)
- Amplification
- Sequencing
- Phylogenetic analysis; multiple sequence
alignment & phylogenetic tree construction.
- MEGA6
Phylogenetic analysis of orientia tsustugamuschi subtypes
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
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
 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.
16s rRNA gene
 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
 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.
 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
 Identification purposes
 Identify crime suspects
 Exonerate persons wrongly accused of crime
 Identify crime and catastrophe victims
 Establish paternity and other family
relationships
 RFLP
 PCR-RFLP
 HLA-DQ
 STR analysis
 Mitochondrial DNA (mtDNA)
 SNP genotyping
 VNTRs (variable number of tandem repeats)
 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.
 Allele specific PCR in HLA typiny
 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.
 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
 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.
************************************

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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.
  • 6. Son-of-son-of-Cycle Baby Blue First device with Peltier heating and cooling system First integrated peltier-based thermal cycler.
  • 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
  • 30.  Taqman probes  Molecular beacons Denaturation Annealing 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
  • 33. Phylogenetic analysis of orientia tsustugamuschi subtypes
  • 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.
  • 44.  Allele specific PCR in HLA typiny
  • 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.

Notas do Editor

  1. 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.
  2. 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.
  3. Mr. cycle- only 3 made- 1- museum of american history, 2. science museum- london, 3. mol.bio dept. univ of southern california.
  4. Roche funds cetus R&D. ROCHE has had the capability to commercialize diagnostic kits world wide.
  5. Sosoc- 2 unit device with base unit and controller. Baby blue- base unit and controller merged
  6. Roche got the right to develop and distribute pcr based diagnostic products by paying royalty to cetus
  7. Inhibitors
  8. K+,NH+,
  9. MICRO, PHARMA, FORENSIC, CANCER, MOLECULAR MEDICINE, PERSONALISED MEDICINE