23 Molecular Pathology Techniques #1 Flashcards
Describe the steps of PCR
- Denaturation: heat to denature -> template strands
- Annealing: cool to allow primers to bind to template strands
- Extension: Heat so Taq polymerase can extend primers, synthesizing new DNA strands
- Denaturation (90-100°C): of the template into single strands // Heat the reaction strongly to denature -> This provides single-stranded template for the next step.
- Annealing (45-65°C): of primers to each original strand for new strand synthesis // Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA
- Extension (72°C): of the new DNA strands from the primers // Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
Name three types of PCR and explain situations in which they should be used
- Endpoint: detection of product
- Allele-specific: Detection of specific mutation
- RT-PCR: Detection of fusion gene products
- qPCR: Quantitation of product
Identify specific situations in which break-apart FISH would be preferred to fusion FISH
Entities in which a single gene pairs with multiple fusion partners (eg EWSR)
Describe the principles behind Sanger sequencing and NGS-based sequencing
Sanger: Single product sequencing by synthesis with fluorescently labeled ddNTPS causing chain termination
NGS: Multiplex, multisource, massively parallel sequencing that uses fragmentation, tagmentation, and immobilization followed by sequencing to do huge amounts of sequencing at once
Describe the principles behind Sanger sequencing and NGS-based sequencing
NGS: Multiplex, multisource, massively parallel sequencing that uses fragmentation, tagmentation, and immobilization followed by sequencing to do huge amounts of sequencing at once
Sanger: Single product sequencing by synthesis with fluorescently labeled ddNTPS causing chain termination
principles behind Sanger sequencing and NGS-based sequencin
Sanger: ? sequencing by synthesis with ? labeled ? causing ?
NGS: Multiplex, multisource, massively parallel sequencing that uses ?, ?, and ? followed by ? to do huge amounts of sequencing at once
Sanger: Single product sequencing by synthesis with fluorescently labeled ddNTPS causing chain termination
NGS: Multiplex, multisource, massively parallel sequencing that uses fragmentation, tagmentation, and immobilization followed by sequencing to do huge amounts of sequencing at once
Four reasons for genetic testing of cancers?
- Diagnosis
- Prognosis
- Therapeutic choice
- Identification of inherited predispositions
What is PCR?
Polymerase Chain Reaction
Canonical amplication-based assay
-fast and inexpensive technique used to “amplify” - copy - small segments of DNA
“molecular photocopying”
What are the three steps of PCR?
- Denaturation (90-100°C): of the template into single strands // Heat the reaction strongly to denature -> This provides single-stranded template for the next step.
- Annealing (45-65°C): of primers to each original strand for new strand synthesis // Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA
- Extension (72°C): of the new DNA strands from the primers // Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
- PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
- Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis
Describe the three steps of PCR:
1. Denaturation
2. Annealing
3. Extension
- Denaturation (90-100°C): of the template into single strands // Heat the reaction strongly to denature -> This provides single-stranded template for the next step.
- Annealing (45-65°C): of primers to each original strand for new strand synthesis // Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA
- Extension (72°C): of the new DNA strands from the primers // Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
- PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
- Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis
Describe the three steps of PCR:
1. Denaturation
2. Annealing
3. Extension
- PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
- Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis
- Denaturation (90-100°C): of the template into single strands // Heat the reaction strongly to denature -> This provides single-stranded template for the next step.
- Annealing (45-65°C): of primers to each original strand for new strand synthesis // Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA
- Extension (72°C): of the new DNA strands from the primers // Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
- PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
- Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis
Describe the three steps of PCR:
1. Denaturation
2. Annealing
3. Extension
- Denaturation (90-100°C): of the template into single strands // Heat the reaction strongly to denature -> This provides single-stranded template for the next step.
-
Annealing (45-65°C): of primers to each original strand for new strand synthesis // Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA
3.Extension (72°C): of the new DNA strands from the primers // Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
- PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
- Taq polymerase can only make DNA if it’s given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis
How does PCR result in amplification?
Amplification occurs as progressive doubling with an exponential and linear phase
- not just the original DNA that’s used as a template each time.
- new DNA that’s made in one round can serve as a template in the next round of DNA synthesis.
- Many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in each round of cycling.
What are the exponential, linear and plateau phases of PCR?
- Exponential phase, no factor is limiting, and the amplification products accumulate at a steady rate.
- [DNA target] may accumulate to a high enough level that one of the PCR reagents will no longer be sufficient to support geometric amplification ->
- Linear phase is the second phase in PCR in which the efficiency declines cycle-to-cycle. Changes in efficiency during linear phase become less and less consistent with increasing cycle number, so the data becomes less and less quantitative
- At some point, reaction components become limiting, and the efficiency of amplification drops and eventually stops; this is the plateau phase. Plateau phase data is not considered quantitative
What are four types of PCR?
- End-point (ie Regular PCR)
- Allele-specific (ARMS)
- Reverse Transcription (RT)
- Realtime/quantitative
Endpoint PCR = Evaluated at endpoint (plateau phase) // Binary detection (Amplified/non-amplified) // FLT3 ITD detection
ARMS = One primer set for mutant allele // One primer set for “normal” allele // Amplification of the mutant allele indicates that the patient harbors the point mutation (JAK2 V617F)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) // One step (TR/PCR in same tube) vs Two step (RT in one tube, PCR in 2nd)
Realtime/quantitative PCR = Formation of amplified product is monitored in each cycle (Ie realtime) // determination of cycle threshold during linear phase allows one to quantitate results using known standards
Describe each of the four PCR types:
Allele-specific PCR (ARMS)
ARMS:
- One primer set for mutant allele //
- One primer set for “normal” allele //
- Amplification of the mutant allele indicates that the patient harbors the point mutation
- based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including
- the transition, transversion and insertion/deletion polymorphism
- eg JAK2 V617F
ARMS = amplification refractory mutation system
Endpoint PCR = Evaluated at endpoint (plateau phase) // Binary detection (Amplified/non-amplified) // FLT3 ITD detection
ARMS = One primer set for mutant allele // One primer set for “normal” allele // Amplification of the mutant allele indicates that the patient harbors the point mutation (JAK2 V617F)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) // One step (TR/PCR in same tube) vs Two step (RT in one tube, PCR in 2nd)
Realtime/quantitative PCR = Formation of amplified product is monitored in each cycle (Ie realtime) // determination of cycle threshold during linear phase allows one to quantitate results using known standards
Describe each of the four PCR types:
1. End-point (ie Regular PCR)
- Allele-specific (ARMS)
- Reverse Transcription (RT)
- Realtime/quantitative
Endpoint PCR =
- Evaluated at endpoint (plateau phase)
- end products are visualized on an agarose gel to determine their size as well as relative quantity
- used for applications such as cloning, sequencing, genotyping and** sequence detection**
- Binary detection (Amplified/non-amplified)
- far less quantitative than real-time PCR—it is used mostly to detect** presence or absence of targets**, but can also be used to estimate relative quantity.
- eg: FLT3 ITD detection
Endpoint PCR = Evaluated at endpoint (plateau phase) // Binary detection (Amplified/non-amplified) // FLT3 ITD detection
ARMS = One primer set for mutant allele // One primer set for “normal” allele // Amplification of the mutant allele indicates that the patient harbors the point mutation (JAK2 V617F)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) // One step (TR/PCR in same tube) vs Two step (RT in one tube, PCR in 2nd)
Realtime/quantitative PCR = Formation of amplified product is monitored in each cycle (Ie realtime) // determination of cycle threshold during linear phase allows one to quantitate results using known standards
Describe each of the four PCR types:
Reverse Transcription (RT)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) //
- One step (TR/PCR in same tube) vs
- Two step (RT in one tube, PCR in 2nd)
- RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here, the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes.
- enable molecular cloning, sequencing or simple detection of RNA
cDNA = complementary DNA
Endpoint PCR = Evaluated at endpoint (plateau phase) // Binary detection (Amplified/non-amplified) // FLT3 ITD detection
ARMS = One primer set for mutant allele // One primer set for “normal” allele // Amplification of the mutant allele indicates that the patient harbors the point mutation (JAK2 V617F)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) // One step (TR/PCR in same tube) vs Two step (RT in one tube, PCR in 2nd)
Realtime/quantitative PCR = Formation of amplified product is monitored in each cycle (Ie realtime) // determination of cycle threshold during linear phase allows one to quantitate results using known standards
Describe each of the four PCR types:
Realtime/quantitative
Realtime/Quantitative = Formation of amplified product is monitored in each cycle (ie realtime) //
- determination of cycle threshold during linear phase allows one to quantitate results using known standards
- In conventional PCR, the amplified DNA product, or amplicon, is detected in an end-point analysis. In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle.
- First, amplification reactions are set up with PCR reagents and unique or custom primers. Reactions are then run in real-time PCR instruments and the collected data is analyzed by proprietary instrument software
Endpoint PCR = Evaluated at endpoint (plateau phase) // Binary detection (Amplified/non-amplified) // FLT3 ITD detection
ARMS = One primer set for mutant allele // One primer set for “normal” allele // Amplification of the mutant allele indicates that the patient harbors the point mutation (JAK2 V617F)
RT = RNA to cDNA before PCR (polymerase only recognizes DNA) // One step (TR/PCR in same tube) vs Two step (RT in one tube, PCR in 2nd)
Realtime/quantitative PCR = Formation of amplified product is monitored in each cycle (Ie realtime) // determination of cycle threshold during linear phase allows one to quantitate results using known standards
Pros and Cons of EndPoint PCR:
Pros:
-Efficient yes/no answers
-Quick and inexpensive
-Easy detection of insertions/deletions via size analysis
Cons:
-Not useful for quantitation
-Does not detect point mutations
-Nonspecific amplification possible
Recall: Endpoint PCR =
- Evaluated at endpoint (plateau phase)
- end products are visualized on an agarose gel to determine their size as well as relative quantity
- used for applications such as cloning, sequencing, genotyping and** sequence detection**
- Binary detection (Amplified/non-amplified)
- far less quantitative than real-time PCR—it is used mostly to detect** presence or absence of targets**, but can also be used to estimate relative quantity.
- eg: FLT3 ITD detection
Example of how Endpoint PCR used in Molecular Pathology:
- Detection of ?
Detection of FLT3 ITD (internal tandem duplication):
Acute myeloid leukemia with a FLT3 internal tandem duplication (FLT3/ITD) mutation is an aggressive hematologic malignancy with a generally poor prognosis
Pros and Cons of Allele-Specific PCR
Pros:
-Simple detection of point mutations
-Heterozygosity testing possible
CONS:
-Only ampifies the specific SNP targeted
-Poor priming
-Non-specific amplication due to GC “clamping”
An example of when Allele-specific PCR would be used?
JAK2 V617F
JAK2 V617F mutation test means that the person tested is likely to have a myeloproliferative neoplasm (MPN). MPN is a group of rare conditions that affect the bone marrow and result in excessive production of red blood cells, white blood cells, or platelets.
Pros and Cons of RT PCR:
Pros:
-Allows detection of ?
-Can be used to detect ?
-Detects ?
-Can be ?
Cons:
-If RT doesn’t work, ? doesn’t work
-Highly dependent on ?
-Highly dependent on pretest knowledge of ?
Pros and Cons of Reverse Transcription PCR:
Pros:
-Allows detection of fusion genes
-Can be used to detect splice variants
-Detects gene expression
-Can be quantitated (qRT-PCR)
Cons:
-If RT doesn’t work, assay doesn’t work
-Highly dependent on RNA stability
-Highly dependent on pretest knowledge of breakpoints
Example of when RT-PCR might be used?
Detect:
RUNX1-RUNX1T1 gene fusion detection
Quantitative real-time reverse transcription polymerase chain reaction testing in neoplasms with known clonal genetic markers can achieve highly sensitive detection of neoplastic cells in blood or bone marrow samples
In this assay, translocation of chromosome 8q22 to 21q22 resulting in fusion of two genes RUNX1 and RUNX1T1 will be evaluated.
Four Principles of FISH:
1. Labelled DNA probe towards ?
2. DNA from tissue is ?
3. Probe ? within the cell
4. Speciman is washed to remove ? and signal is generated via ?
Fluorescence In Situ Hyridization
Four Principles of FISH:
1. Labelled DNA probe towards region of interest
2. DNA from tissue is denatured and hybridized to probe
3. Probe spatially labels DNA within the cell
4. Speciman is washed to remove nonspecific binding and signal is generated via fluorescent tags on the probe
Fluorescence In Situ Hyridization
What is Enumeration FISH? An example of when it is used?
Enumeration probes are chromosome specific sequences generated from highly repeated human satellite DNA located in the centromeric, pericentromeric or heterochromatic regions of each chromosome
- HER2 copy number
What is Break-Apart FISH?
An Example of when it is used?
- Break-apart probes are designed to detect translocations, the most common genetic abnormality exhibited in cancer cells.
- They’re designed to flank either side of a gene, so that in the event of a translocation, the two colors will split.
- Eg: EWSR (22q12) translocation
What is Fusion FISH?
An Example of when it is used?
- widespread gene fusions underlie many cancers
- probe two segments of the fusion transcripts with differently colored probe sets, and then image the cells in the corresponding fluorescence channels of a fluorescence microscope.
- Diffraction-limited spots visible in both channels correspond to individual fusion transcripts. These spots are seen only in the cancerous cells, and serve as definitive markers of these cells.
- This method not only permits the detection of individual fusion transcript molecules, it provides an explicit count of the number of fusion transcripts in each cell.
What is Sanger Sequencing:
- ? or ? sequencing
- “Sequencing by ?”
- for determining ?
- ? PCR w/ added ?
- Result?
What is Sanger Sequencing:
- Dideoxynucleotide or dye termination sequencing
- Sequencing by synthesis
- “chain termination method”, is a method for determining the nucleotide sequence of DNA.
- Chain termination PCR w/ ddNTPs
- In chain-termination PCR, the user mixes a low ratio of chain-terminating ddNTPs in with the normal dNTPs in the PCR reaction.
- ddNTPs lack the 3’-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases.
- The result of chain-termination PCR is many oligonucleotide copies of the DNA sequence of interest, terminated at a random lengths (n) by 5’-ddNTPs
What is NGS
Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed.
The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA
Basic Methods of NGS:
- All NGS devices follow the same basic principles that allow for ?
- The “-ations”:
- (5)
Basic Methods of NGS:
- All NGS devices follow the same basic principles that allow for massive parallel sequencing
- The “-ations”:
- Fragmentation
- Tagmentation
- Immobolization
- Detection (sequencing)
- Computation (“pipeline”)
NGS
Library Generation:
- Involves ? and ?
- Creation of suitable ? for sequencing
- ? creates DNA pieces small enough to be efficiently sequenced)
Library Generation:
- Involves Fragmentation and Tagmentation
- Creation of suitable dsDNA specimen for sequencing
- Fragmentation creates DNA pieces small enough to be efficiently sequenced)
NGS - Library generation
What is Tagmentation?
Tagmentation:
- Create DNA fragments with tags for unique ID, for universal priming and for immobilization
What is Computation in NGS?
- The “?”
- The bioinformatic process of taking raw ? and generating a list of “?” present in the sequenced sample
- The “PIPELINE”
- The bioinformatic process of taking raw sequence data and generating a list of “variants” present in the sequenced sample
Key NGS Concepts:
- Large (but finite) number of ? are available
- ? are of a set size (typically 100-300bp)
- Amount of sequence is divided among ?
- The ability to find a ? is based on two major concepts:
1. ?
2. ?
- Large (but finite) number of sequence reads are available
- Reads are of a set size (typically 100-300bp)
- Amount of sequence is divided among all regions analyzed
- The ability to find a mutation is based on two major concepts:
1. Depth of coverage
2. Breadth of coverage
What is Depth of Coverage?
- Number of times a specific base or sequence has been “read” during the assay
- Helps separate random error from true mutation
- Also directly correlates to test sensitivity
What is Uniformity of Coverage?
- The distribution of ? across the ?
- Important for ?
- The distribution of reads across the regions of interest
- Important for determination of copy number variation
Depth and Sensitivity
- Low Level mutations are accurately detected with greater than 500 read depth
- the higher the number of aligned sequence reads, the higher the confidence to the base call at a particular position, regardless of whether the base call is the same as the reference base or is mutated (1). In other words, individual sequencing error reads are statistically irrelevant when they are outnumbered by correct read
