Unit 7 - Techniques and Diagnostics Flashcards
DNA diagnostic tests
PCR
Reverse Transcriptase PCR (RT-PCR)
real time PCR
DNA sequencing and Next Generation DNA sequencing
DNA microarrays
what is PCR named after
Taq DNA Polymerase from thermus aquaticus that is used to amplify a piece of DNA by in vitro enzymatic replication
MOA of PCR
as PCR progresses, the DNA generated is itself used as a template for replication
This sets in motion a chain reaction in which the DNA template is exponentially amplified
With PCR it is possible to amplify, very specifically, a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece
what is needed for PCR
DNA template that contains the DNA region (target) to be amplified
Two primers, forward and reverse, which are complementary to the DNA regions at the 5’ or 3’ ends of the DNA region
Taq polymerase with a temperature optimum at around 70°C
Deoxynucleotide triphosphates (dNTPs) the building blocks from which the DNA polymerases synthesizes a new DNA strand
Buffer solution which contains Mg2+ , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
thermal cycling
alternately heating and cooling the PCR sample to a defined series of temperature steps
3 steps of PCR procedure
- denaturation of the template DNA at 94°C
- Annealing of the single stranded primers at 55-65°C
- extension of the annealed primers by addition of nucleotides by base pairing to the template DNA at 72°C
1st cycle
after 1 cycle of synthesis the rxn mixture is again heated to dissociate the DNA strands and cooled to re-anneal the DNA and primers
primers are extended again
2nd cycle
2 newly synthesised, single stranded chains are precisely the length of the DNA between the 5’ ends of the primers
3rd cycle
2 double-stranded DNA molecules that exactly match the target sequence are produced
what changes with each cycle
the number of DNA strands, whose 5’ to 3’ ends are defined by the ends of the primers, increases exponentially
As a result, the desired DNA is preferentially replicated until after 20-30 cycles, it makes up most of the DNA in the tube
(PCR products are visualized by agarose gel electrophoresis)
what is Agarose Gel Electrophoresis
how does it work
agarose is a polysaccharide which acts as a molecular sieve
An electric current is applied across the gel
DNA is negatively charged and is attracted to the positive anode
The DNA is separated based on size – shorter molecules move faster through gel than longer
Gel contains ethidium bromide dye (or SYBR Green) to allow DNA to be seen under ultraviolet light
advantages of PCR
time taken to amplify sufficient amounts of the target sequence
a single molecule of the target sequence can be amplified to 109 copies in 1.5-6 hours - In contrast, it takes several days to weeks to produce similar levels using cell-based approaches (i.e. plasmid vectors and host bacterial cells)
Sensitivity
A single copy of the target DNA sequence can be amplified rapidly to usable concentrations (e.g. can be visualized on a gel), hence the usefulness of PCR in forensic science
Robustness
PCR can be used to amplify target gene sequence information from partially degraded DNA samples or from tissues that have been formalin-fixed on slides
disadvantages of PCR
1. Prior sequence knowledge is essential
forward and reverse primers are designed from known DNA sequence data
2. Limited size range of PCR products
PCR products are generally 200-100 bases in length (most accurate/reproducible range), although products of up to 5 kb have been amplified (rare)
3. DNA Replication may be inaccurate
in a standard PCR reaction using an ordinary Taq Polymerase preparation, as much as 40% of the products will contain some error in the nucleotide sequence
4. Contamination/False Positives
Contamination from the operator or previous PCR reactions can lead to false positives
5 medical applications of PCR
- Genetic testing for e.g. carriers of cystic fibrosis etc
- Pre-natal testing - disease mutation DNA samples can be obtained by amniocentesis or chorionic villus sampling
- Pre-implantation genetic diagnosis where individual cells of a developing embryo are tested for mutations
- Tissue typing for organ transplantation - proposal to replace the traditional antibody-based tests for blood type with PCR-based tests
- Diagnosis of Infectious Disease e.g. Human Immunodeficiency Virus or Human Papilloma Virus or Hepatitis
how is PCR used in cancer diagnostics
Many forms of cancer involve alterations to genes e.g. proto-oncogenes are mutated to oncogenes
by using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient
PCR and the bcr-abl oncogene
how is bcr-abl formed
The bcr-abl oncogene is the result of a translocation of DNA sequences from human chr9 to chr22 (Philadelphia Chromosome)
PCR can be used to detect the bcr-abl oncogene and determine which variant of the gene is present
→ Produces a new fusion protein from BCR and ABL genetic sequences
This translocation and the bcr-abl tyrosine kinase are present in 95% of chronic myelogenous leukemia (CML)
applications of RT-PCR
Reverse transcription polymerase chain reaction is widely used in the diagnosis of genetic diseases
semi-quantitatively, in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression
to determine risk of re-occurence of breast cancer in patients with stage 1 or 2 node-negative breast cancer
(limited - only a number of genes can be amplified)
Oncotype-DX
RT-PCR-based assay performed on RNA extracted from paraffin-embedded tumour tissue
determines the level of expression in 21 genes, 16 of which are cancer-related genes and 5 are control reference genes
MOA of real time/quantitative PCR
monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR
Fluorescent label e.g. SYBR Green is added to the DNA during amplification process, and detected by the Real Time PCR Machine
Real-time PCR can be used quantitatively (quantitative real-time PCR) and semiquantitatively (i.e., above/below a certain amount of DNA molecules) (semiquantitative real-time PCR)
Real time PCR – COVID-19 Testing
uses for Chain Termination (Sanger) DNA Sequencing
genome projects - easily automated
(other methods include Chemical Degradation Method and Pyrosequencing)
steps in the Chain Termination (Sanger) DNA Sequencing
First step – annealing of a short oligonucleotide primer to the same position on each DNA molecule
Acts a primer for the synthesis of new DNA strand complementary to the template
The strand synthesis reaction is catalyzed by the enzyme DNA Polymerase
Requires four deoxyribonucleotides triphosphates (dNTPS) – dATP, dTTP, dCTP and dGTP
Also, a small amount of terminating nucleotides, dideoxynucleotide triphosphates (ddNTPS) – ddATP, ddTTP, ddCTP and ddGTP are required to produce DNA fragments
Each dideoxynucleotide is labeled with a different fluorescent marker
Polymerase enzyme does not discriminate between deoxy- and dideoxynucleotides
Once incorporated a dideoxynucleotide blocks further strand elongation because it lacks the 3’-hydroxyl group needed to form the connection with the next nucleotide
Because normal deoxynucleotides are present in larger amounts than the dideoxynucleotides, the strand synthesis does not always terminate close to the primer
The result is a set of new molecules, all of different lengths ending in a dideoxynucleotide which indicates a nucleotide A,C,G, or T that is present at the equivalent position in the template
main events of Chain Termination (Sanger) DNA Sequencing
Incorporation of ddATP results in chains that are terminated opposite Ts in the template – generated a family of ‘A’ terminated molecules
Incorporation of other ddNTPs generates ‘C’, ‘G’ and ‘T’ families
Each dideoxynucleotide is labeled with a different fluorophore
what happens during electrophoresis in Sanger DNA Sequencing
how is the information interpreted
During electrophoresis, the labeled molecules move past a fluorescence detector, which identifies which dideoxynucleotide is present in each band
The information is passed on to an imaging system
The DNA sequence is represented by a series of peaks, one for each nucleotide position
The sequence can be printed out or entered directly into a storage device for future analysis
Automated sequencers with 96 capillaries in parallel – average of 750 bp per experiment – 864 kb can be generated per machine per day but requires 24 hour support, robotic devices to prepare sequencing reactions and load sequencers, to generate sequence of an entire genome in weeks
Next Generation Sequencing Technologies (NGST) types
Roche 454 Sequencing
Applied Biosystems/ SOLiD
Illumina Genome Analyzer
Helicos
MOA of Roche - 454 Sequencing
Step 1: Preparation of an adapter ligated single stranded DNA library i.e. DNA fragments of the entire genome are ligated to adapters to which PCR primers are designed
Step 2: Individual DNA fragments are attached to beads via the adapters
Step 3: The DNA fragment on each bead is amplified by emulsion PCR (EmPCR) i.e. the beads are suspended in an oil emulsion which includes PCR regents including primers designed from the adapter sequences
Step 4: After amplification by PCR, the individual beads are distributed into the wells of a PicoTiter Plate™
Step 5: The DNA fragments on each bead are sequenced by pyrosequencing using a 454 Sequencer
Pyrosequencing (put in steps)
how has NGST helped in clinical practice
helped the discovery of DNA sequence variants with clinical significance
use of DNA sequence of diagnostic markers is entering into clinical practice for the detection of DNA sequence variants or small insertions or deletions in genes
somatic changes in DNA from tumour tissue but not present in normal tissue the same person
Oncogenic DNA sequence variants are useful diagnostic biomarkers and have provided specific molecular targets for cancer therapies e.g. Pancreatic Cancer Model
interaction between genes and their product
1000s of genes and their product i.e. RNA and proteins in a given organism function in a complicated and harmonious way
DNA microarray (DNA chip or gene chip) analyses
an entire genome in one experiment
what can the DNA microarray be used to study
gene expression and transcription rates of the genome in vivo - The genes that are transcribed at any particular time are known as the transcriptome
transcriptome
the genes that are transcribed at any particular time (DNA microarrays)
principle behind the microarray
the placement of specific nucleotide sequences in an ordered array
These base-pair with complementary sequences of DNA or RNA that have been labelled with fluorescent markers of different colours
The locations of the binding occurred and the colours observed are then used to quantify the amount of DNA or RNA bound
Microarrays are manufactured by high-speed robotics which can put thousands of samples on a glass slide with an area of 1cm2
The diameter of an individual sample can be 200µm or less
what could a microarray be used to determine
if a new anti-cancer drug would be harmful to the liver
microarray step 1
constructed so that it has a single-stranded DNA representing thousands of different genes, each applied to a specific spot on the microarray chip
microarray step 2
different populations of liver cells (e.g.) are collected, one treated with the potential drug and the other untreated
The mRNA being transcribed in these cells is then isolated
microarray step 3
the mRNA is converted to cDNA using reverse transcriptase enzyme
Green fluorescent labels are added to the cDNA from the untreated cells and red fluorescent labels are added to the cDNA from the treated cells
microarray step 4
the labelled cDNAs are added to the DNA chip
The cDNAs bind to the chip if they find their complementary sequences in the singlestranded DNAs loaded onto the chip
microarray step 5
the chip is scanned and a computer analyses the fluorescence
The results appear a as a series of coloured dots
A red dot indicates a DNA sequence that bound to cDNA from the treated cells
This indicates an mRNA that was expressed in treated cells
A green dot indicates RNA produced in untreated cells but not treated cells
A yellow dot would indicate that mRNA was produced equally well in treated and untreated cells
Blank spaces indicate spaces on the DNA chip for which no mRNA was produced in either situation
microarray step 6
To answer the question of whether the potential drug is toxic to liver cell, the results from the microarray would be compared to controls run with liver cells and drugs known to be toxic verses those known to be non-toxic
overview of microarray
how can microarrays be used in diagnosis
e.g. to scan cells from cancer patients and correlate microarray patterns with prognosis
The four different patterns are compared to the percentage of patients who develop metatases
Information like this would be crucial in the treatment of cancer – helps to choose correct treatment for each patient
Also, to determine the type of leukaemia or lymphoma
MammaPrint
Biomarkers of breast cancer subtypes were identified using gene expression patterns in microarrays – using hierarchical clustering of gene expression
Large gene sets (70 genes) were able to identify five subtypes of breast cancer including basal-like (often seen in carriers of BRCA1 mutations), HER2-overexpressing subtype, two types of luminal cells and normal tissue-like subgroup
what does whole genome expression profiling allow for
the opportunity to identify features of the transcriptome that indicated good or poor survival
protein microarray
what is its advantage
A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale
Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel
how does protein microarray work
The chip consists of a support surface such as a glass slide, nitrocellulose (nc) membrane, bead, or microtitre plate, to which an array of capture proteins is bound
Probe molecules, typically labeled with a fluorescent dye, are added to the array
Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner
Fluorescently labelled antibodies are the added and the microarray scanned
advantages of protein microarray
uses
Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents
E.g. antibodies to known diseases can be bound to the microarray
If a patient has a particular disease, proteins specific to that disease bind to the appropriate antibodies
scan to detect anthrax
what is protein microarray limited by
availability of antibodies
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE)
uses
A widely used technique to determine the relative molecular mass of a protein and characterize the proteins found in a sample e.g. blood serum
overview of MOA - SDS PAGE
electrophoresis of the mixture of proteins through a gel matrix that is formed by polymerization of acrylamide and bisacrylamide between a pair of glass plates
PAGE of proteins is typically carried out using protein mixtures that have been denatured by heating in the presence of a detergent - SDS
SDS is a negatively charged molecule that becomes covalently linked to proteins along their length upon exposure to heat, as well as denaturing the protein, SDS also imparts a negative charge in proportion to its length
Upon introduction of the protein sample to the gel and the application of a vertical electric field from the top of the gel to the bottom, proteins are repelled by the negative pole (cathode) and migrate towards the positive pole (anode)
Due to the molecular sieving effect of the gel matrix, proteins within the mixture become resolved into discrete zones (bands) with the smallest proteins moving furthest through the gel (A protein ladder is also applied to the gel for reference)
the separated proteins are visualised by staining with the dye Comassie Blue
Western Blotting - why is it needed
Antibodies are relatively large molecules and cannot penetrate the gel matrix, so the gel is blotted onto a positively charged membrane that traps the charged proteins and immobilizes them on the surface of the membrane
MOA of Western Blotting
achieved by again applying an electric field to the gel to drive the proteins horizontally out of the gel onto the blotting membrane e.g. polyvinylidene difluoride (PVDF) or nitrocellulose-based membranes
The blot can then be probed with antibodies directed against the protein (antigen) (Ag) of interest
visualisation of Ag-Ab complexes (form on the band containing the protein recognised by the antibody)
If the protein of interest was bound by a radioactive antibody, its position can be determined by exposing the membrane to a sheet of x-ray film, a procedure termed autoradiography
However, the most generally used detection procedures employ enzyme-linked antibodies against the antigen - After binding of enzyme-antibody conjugate, addition of a chromogenic substrate that produces a highly coloured and insoluble product causes the appearance of a coloured band at the site of the target antigen
Even greater sensitivity is achieved if a chemiluminescent compound with suitable enhancing agents is used to produce light at the antigen site
applications of Western Blotting
The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample
A Western blot is also used as the definitive test for prion diseases e.g. CreutzfeldJakob Disease (CJD) and Bovine spongiform encephalopathy (BSE) ‘mad cow disease’)
Lyme disease caused by the bacterium Borrelia burgdorferi (transmitted by tick bite) – test for IgM & IgD antibodies (ELISA) followed by Western blotting to confirm
Western blot is the confirmatory test for Hepatitis B infection
how does Hepatitis B infection work
how can it be detected
the genome of hepatitis B virus (HBV) encodes the so-called “X” protein (HBxAg)
HBxAg is a common marker in the livers of carriers
use of western blotting in cancer research
E.g. Cell Signaling Technology sells an ‘Oncogene and Tumour Suppressor Sampler Kit’ containing primary antibodies against eight proteins including BRCA1, HER2, p53, phospho-estrogen receptor etc
Enzyme-Linked Immunosorbent Assay (ELISA)
what is it used for
ELISAs are designed for detecting and quantifying substances such as
➢ peptides
➢ proteins
➢ antibodies
➢ hormones
➢ cytokines
➢ drugs of abuse and their metabolites
name the 3 types of ELISA
- indirect
- sandwich
- competitive
explain Indrect ELISA
involves coating of antigen onto microtitre plates, followed by incubation with a specific antibody
the binding antibody or an appropriate secondary antibody is conjugated to an enzyme that typically catalyses formation of a coloured product
When certain enzymes e.g. peroxidase react with appropriate substrates such as 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), or 3,3’,5,5’-Tetramethylbenzidine (TMB), they can result in changes in color, which can be used as a signal
Color formation is monitored spectophotometrically and related to concentration of antigen by calibration to a standard curve
The technique is used to measure antibodies e.g. antibodies against HIV or an anticancer antibody
applications of indirect ELISA
explain the MOA of its application
HIV - Indirect ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS
The confirmatory tests is Western Blot and PCR
In this assay, recombinant envelope and core proteins of HIV are absorbed as solidphase antigens to microtitre wells
Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins
Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection
sandwich ELISA
Antigen can be detected or measured by a sandwich ELISA
In this technique, the antibody (rather than antigen) is immobilized on a microtitre well
A sample containing antigen is added and allowed to react with the immobilised antibody
After the well is washed, a second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen
After any free second antibody is removed by washing, substrate is added, and the coloured reaction product is measured
Miniturized version of this assay used in antibody microarrays
applications of sandwich ELISA
used to measure large antigens
pregnancy testing - Human Chorionic Gonadotrophin (hCG)
Mucus glycoproteins (mucins) are frequently detectable in the serum of patients with Pancreatic cancer - Muc 1 core protein has a m.w. of 120,000-225,000 (250,000- 500,000 in glycolylated forms)
CD44 ( m.w. 85,000) detection in Bladder Cancer - Also, CD44 variant proteins (m.w. 150,000 -200,000)
Onko-Sure™ test detects Fibrin Degradation Products (FDP) – diagnostic for colorectal cancer, breast, lung, ovarian etc
competitive ELISA procedure
antibody is first incubated in solution with a sample containing antigen
The antigen-antibody mixture is then added to antigen-coated microtitre well
The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well
Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well, as in indirect ELISA
In competitive ELISA, however, the higher the concentration of antigen in the original sample, the lower the absorbance
applications of competitive ELISA
used to measure small antigens e.g. steroids (oestradiol 17β), drugs, peptides (insulin)
used to measure several cancer biomarkers including Osteopontin (prostate cancer),
Bone Sialoprotein (BSP) (colon, breast, prostate, lung cancers),
Cluster of Differentiation 147 (CD147) (aka Basigin & extracellular matrix metalloproteinase inducer (EMMPRIN) - found on tumour cell surfaces and promoting tumour invasion – associated with mouth and throat cancer
