Polymerase chain reaction technique Flashcards
Key points:
Complementarity: base pairs form only between G and C, A and T or A and U
Denaturation: the separation of a DNA duplex into single-stranded DNA
Hybridisation: joining a nucleic acid probe to its complementarity sequences (may be DNA:DNA or DNA:RNA)
Replication: a template is used to duplicate the genetic sequence by enzymatic addition of complementary bases
Key points
Complementarity: base pairs form only between G and C, A and T or A and U
Denaturation: the separation of a DNA duplex into single-stranded DNA
Hybridisation: joining a nucleic acid probe to its complementarity sequences (may be DNA:DNA or DNA:RNA)
Replication: a template is used to duplicate the genetic sequence by enzymatic addition of complementary bases
Real time - PCR technique
RNA extracted from FFPE tumour blocks.
Complementary DNA (cDNA) is syn............ Pri......., Poly........, Nucle....... required.
RT-PCR is performed – e.g. gene ex…….. assays or gene-fusions
On graph
lower Cq (cycle) means larger abundant material
higher cq means scarce starting material
Real time - PCR technique
RNA extracted from FFPE tumour blocks.
Complementary DNA (cDNA) is synthesised.
Primers, Polymerase, Nucleotides required.
RT-PCR is performed – e.g. gene expression assays or gene-fusions
On graph
lower Cq (cycle) means larger abundant material
higher cq means scarce starting material
PCR
Most basic of molecular testing, sequences of ………. in …….. to identify alterations (e.g. single nu………. vari………, del………, inse…….). Common principle is p……… complementary to particular sequences of interest are added to the DNA sample. Nucleotides and DNA poly…….. are added and the whole solution unde……cycles of heating and cooling.
Replication occurs with each cycle and there is an exp………. increase of the number of DN…… strands in the solution.
F……. tissue is used to extract DNA and/or …… using purifi…….. kits.
Used for detection of genetic abnormalities and in companion diag,,,,,,,, (targeted drugs).
Examples of application: EGFR1 in non-small cell lung cancer; BRAF in melanoma; KRAS and NRAS in colorectal cancer.
Most basic of molecular testing, sequences of bases in DNA to identify alterations (e.g. single nucleotide variants, deletions, insertions). Common principle is primers complementary to particular sequences of interest are added to the DNA sample. Nucleotides and DNA polymerase are added and the whole solution undergoes cycles of heating and cooling.
Replication occurs with each cycle and there is an exponential increase of the number of DNA strands in the solution.
FFPE tissue is used to extract DNA and/or RNA using purification kits.
Used for detection of genetic abnormalities and in companion diagnostics (targeted drugs).
Examples of application: EGFR1 in non-small cell lung cancer; BRAF in melanoma; KRAS and NRAS in colorectal cancer.
Uses of PCR
PCR is an extremely powerful technique which has adopted central importance in molecular pathology laboratories because of its versatility in testing for an exceptionally broad range of ……… abnormalities. The advent of targeted therapies for certain cancers has immensely increased the importance of PCR-based technologies.
Pharmaceutical companies develop targeted drugs alongside a companion diagnostic. The companion diagnostic is an assay which identifies a limited number of sp…….. genes shown in trials to predict response to the drug in question. Even within the same gene, different dia………. identify different m………. which have been validated for that specific drug. Indeed, using a different assay to that employed in trials for a particular drug can generate con……. because it may identify mut…….. for which the drug has not sp……. been tested.
PCR is an extremely powerful technique which has adopted central importance in molecular pathology laboratories because of its versatility in testing for an exceptionally broad range of genetic abnormalities. The advent of targeted therapies for certain cancers has immensely increased the importance of PCR-based technologies.
Pharmaceutical companies develop targeted drugs alongside a companion diagnostic. The companion diagnostic is an assay which identifies a limited number of alterations in specific genes shown in trials to predict response to the drug in question. Even within the same gene, different diagnostics identify different mutations which have been validated for that specific drug. Indeed, using a different assay to that employed in trials for a particular drug can generate confusion because it may identify mutations for which the drug has not specifically been tested.
EGFR! in non small cell lung caner (NSCLC)
- A proportion of NSCLCs bear activating mutations in the EGFR1 ……… which lead to uncontrolled cell ………… and tumour development.
A number of specific mutations in EGFR1 have been associated with good and sometimes dramatic responses to ………. kinase inh……….. (TKIs).
Only a limited number of mutations have been clinically validated and all are located in hot-spots distributed across exons ….to …..
The drug from Astra Zeneca has been validated with the Therascreen kit and the drug from Roche has been validated with the COBAS kit.
Both assays target approximately 30 mutations within the four exons of interest.
EGFR! in non small cell lung caner (NSCLC)
- A proportion of NSCLCs bear activating mutations in the EGFR1 gene which lead to uncontrolled cell proliferation and tumour development.
A number of specific mutations in EGFR1 have been associated with good and sometimes dramatic responses to tyrosine kinase inhibitors (TKIs).
Only a limited number of mutations have been clinically validated and all are located in hot-spots distributed across exons 18 to 21.
The drug from Astra Zeneca has been validated with the Therascreen kit and the drug from Roche has been validated with the COBAS kit.
BRAF in melanoma
Mutations in the BRAF gene are associated with good responses to anti-BRAF therapy in …………
Two drugs are currently licensed for this purpose, one from Ro…. and the other from Bo……….
Amongst all the mutations which can possibly occur in BRAF, only a limited number have been shown to have clinical value, i.e. are clinically validated.
……. techniques targeted specifically against these mutation hot-spots must demonstrate the presence of a mutation before anti-BRAF therapy can be commenced.
BRAF in melanoma
Mutations in the BRAF gene are associated with good responses to anti-BRAF therapy in melanoma.
Two drugs are currently licensed for this purpose, one from Roche and the other from Boehringer.
Amongst all the mutations which can possibly occur in BRAF, only a limited number have been shown to have clinical value, i.e. are clinically validated.
PCR techniques targeted specifically against these mutation hot-spots must demonstrate the presence of a mutation before anti-BRAF therapy can be commenced.
KRAS and NRAS in colorectal cancer
It is known that a proportion of patients with advanced colorectal cancer will respond to treatment with anti-EGFR1 mo……….. antibodies and two such drugs are currently available for this purpose.
However, it has been shown that the presence of either a KR……. or N….. mutation in the tumour reliably predicts fail…… of response to these drugs and so testing for mutations in these genes is mandatory prior to prescription.
In this way, it is hoped that patients will be spared treatment with drugs which have no beneficial effect.
KRAS and NRAS in colorectal cancer
It is known that a proportion of patients with advanced colorectal cancer will respond to treatment with anti-EGFR1 monoclonal antibodies and two such drugs are currently available for this purpose.
However, it has been shown that the presence of either a KRAS or NRAS mutation in the tumour reliably predicts failure of response to these drugs and so testing for mutations in these genes is mandatory prior to prescription.
In this way, it is hoped that patients will be spared treatment with drugs which have no beneficial effect.
PCR platforms examples
Specimen
FFPE, blood, urine, sputum ++
Cartridge
All reagents for sample prep &PCR
Idulla Platform
Sample in result out
actioable clinical information allowing personalised treatment
One of the biggest challenges in oncology biomarker testing is the ability to obtain samples of sufficient size and quality. With the Idylla system only a minimal amount of sample is needed
1x 5 μm FFPE tissue section -> 50-600 mm² 1x 10 μm FFPE tissue section -> 25-300 mm² Neoplastic cells ≥10% - if less, macrodissection is required
Specimen
FFPE, blood, urine, sputum ++
Cartridge
All reagents for sample prep &PCR
Idulla Platform
Sample in result out
actioable clinical information allowing personalised treatment
One of the biggest challenges in oncology biomarker testing is the ability to obtain samples of sufficient size and quality. With the Idylla system only a minimal amount of sample is needed
1x 5 μm FFPE tissue section -> 50-600 mm² 1x 10 μm FFPE tissue section -> 25-300 mm² Neoplastic cells ≥10% - if less, macrodissection is required
IDYLAA mutation tests examples
The fully automated Idylla KRAS Mutation Test covers 21 KRAS mutations in exons 2,3 and 4 showing an excellent concordance to reference methods of >95%. Comparison with various other KRAS detecting technologies revealed a superior performance of the Idylla KRAS Mutation Test in terms of ease of use, turnaround time, and hands-on time while demonstrating superior levels of sensitivity.
The fully automated Idylla ………. Mutation Test covers BRAF V600E, E2, D, K, R and M mutation with a sensitivity of 1% of mutant in wild type background in FF…….samples with an excellent concordance to reference methods of >95%.
The fully automated Idylla EGFR Mutation Test covers …. mutations in exons 18–…..in a single cartridge using only … FFPE tissue section from metastatic NS….. showing a high concordance of >95% compared with reference methods.
The fully automated Idylla N……-BRAF Mutation Test covers 18 mutations in NRAS exons 2,3 and 4 as well as 5 mutations in BRAF codon 600 showing an excellent co……… to reference methods of >99%.
The fully automated Idylla KRAS Mutation Test covers 21 KRAS mutations in exons 2,3 and 4 showing an excellent concordance to reference methods of >95%. Comparison with various other KRAS detecting technologies revealed a superior performance of the Idylla KRAS Mutation Test in terms of ease of use, turnaround time, and hands-on time while demonstrating superior levels of sensitivity.
The fully automated Idylla BRAF Mutation Test covers BRAF V600E, E2, D, K, R and M mutation with a sensitivity of 1% of mutant in wild type background in FFPE samples with an excellent concordance to reference methods of >95%.
The fully automated Idylla EGFR Mutation Test covers 51 mutations in exons 18–21 in a single cartridge using only 1 FFPE tissue section from metastatic NSCLC showing a high concordance of >95% compared with reference methods.
The fully automated Idylla NRAS-BRAF Mutation Test covers 18 mutations in NRAS exons 2,3 and 4 as well as 5 mutations in BRAF codon 600 showing an excellent concordance to reference methods of >99%.
