Unit 7 - Techniques and Diagnostics

Question 1

DNA diagnostic tests

Answer 1

PCR

Reverse Transcriptase PCR (RT-PCR)

real time PCR
DNA sequencing and Next Generation DNA sequencing

DNA microarrays

Question 2

what is PCR named after

Answer 2

Taq DNA Polymerase from thermus aquaticus that is used to amplify a piece of DNA by in vitro enzymatic replication

Question 3

MOA of PCR

Answer 3

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

Question 4

what is needed for PCR

Answer 4

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

Question 5

thermal cycling

Answer 5

alternately heating and cooling the PCR sample to a defined series of temperature steps

Question 6

3 steps of PCR procedure

Answer 6

1. denaturation of the template DNA at 94°C
2. Annealing of the single stranded primers at 55-65°C
3. extension of the annealed primers by addition of nucleotides by base pairing to the template DNA at 72°C

Question 8

1st cycle

Answer 8

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

Question 9

2nd cycle

Answer 9

2 newly synthesised, single stranded chains are precisely the length of the DNA between the 5’ ends of the primers

Question 10

3rd cycle

Answer 10

2 double-stranded DNA molecules that exactly match the target sequence are produced

Question 11

what changes with each cycle

Answer 11

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)

Question 12

what is Agarose Gel Electrophoresis

how does it work

Answer 12

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

Question 13

advantages of PCR

Answer 13

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

Question 14

disadvantages of PCR

Answer 14

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

Question 15

5 medical applications of PCR

Answer 15

1. Genetic testing for e.g. carriers of cystic fibrosis etc
2. Pre-natal testing - disease mutation DNA samples can be obtained by amniocentesis or chorionic villus sampling
3. Pre-implantation genetic diagnosis where individual cells of a developing embryo are tested for mutations
4. Tissue typing for organ transplantation - proposal to replace the traditional antibody-based tests for blood type with PCR-based tests
5. Diagnosis of Infectious Disease e.g. Human Immunodeficiency Virus or Human Papilloma Virus or Hepatitis

Question 16

how is PCR used in cancer diagnostics

Answer 16

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

Question 17

PCR and the bcr-abl oncogene

how is bcr-abl formed

Answer 17

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)

Question 18

applications of RT-PCR

Answer 18

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)

Question 20

Oncotype-DX

Answer 20

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

Question 21

MOA of real time/quantitative PCR

Answer 21

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

Question 22

uses for Chain Termination (Sanger) DNA Sequencing

Answer 22

genome projects - easily automated

(other methods include Chemical Degradation Method and Pyrosequencing)

Question 23

steps in the Chain Termination (Sanger) DNA Sequencing

Answer 23

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

Question 24

main events of Chain Termination (Sanger) DNA Sequencing

Answer 24

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

Question 25

what happens during electrophoresis in Sanger DNA Sequencing how is the information interpreted

Answer 25

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

Question 26

Next Generation Sequencing Technologies (NGST) types

Answer 26

Roche 454 Sequencing Applied Biosystems/ SOLiD Illumina Genome Analyzer Helicos

Question 27

MOA of Roche - 454 Sequencing

Answer 27

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

Question 28

Pyrosequencing (put in steps)

Answer 28

Question 29

how has NGST helped in clinical practice

Answer 29

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

Question 30

interaction between genes and their product

Answer 30

1000s of genes and their product i.e. RNA and proteins in a given organism function in a complicated and harmonious way

Question 31

DNA microarray (DNA chip or gene chip) analyses

Answer 31

an entire genome in one experiment

Question 32

what can the DNA microarray be used to study

Answer 32

gene expression and transcription rates of the genome in vivo - The genes that are transcribed at any particular time are known as the transcriptome

Question 33

transcriptome

Answer 33

the genes that are transcribed at any particular time (DNA microarrays)

Question 34

principle behind the microarray

Answer 34

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

Question 35

what could a microarray be used to determine

Answer 35

if a new anti-cancer drug would be harmful to the liver

Question 36

microarray step 1

Answer 36

constructed so that it has a single-stranded DNA representing thousands of different genes, each applied to a specific spot on the microarray chip

Question 37

microarray step 2

Answer 37

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

Question 38

microarray step 3

Answer 38

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

Question 39

microarray step 4

Answer 39

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

Question 40

microarray step 5

Answer 40

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

Question 41

microarray step 6

Answer 41

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

Question 42

overview of microarray

Answer 42

Question 43

how can microarrays be used in diagnosis

Answer 43

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

Question 44

MammaPrint

Answer 44

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

Question 45

what does whole genome expression profiling allow for

Answer 45

the opportunity to identify features of the transcriptome that indicated good or poor survival

Question 46

protein microarray what is its advantage

Answer 46

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

Question 47

how does protein microarray work

Answer 47

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

Question 48

advantages of protein microarray uses

Answer 48

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

Question 49

scan to detect anthrax

Answer 49

Question 50

what is protein microarray limited by

Answer 50

availability of antibodies

Question 51

**Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE)** uses

Answer 51

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

Question 52

overview of MOA - SDS PAGE

Answer 52

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

Question 53

Western Blotting - why is it needed

Answer 53

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

Question 55

MOA of Western Blotting

Answer 55

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

Question 56

visualisation of Ag-Ab complexes (form on the band containing the protein recognised by the antibody)

Answer 56

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

Question 57

applications of Western Blotting

Answer 57

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

Question 58

how does Hepatitis B infection work how can it be detected

Answer 58

the genome of hepatitis B virus (HBV) encodes the so-called "X" protein (HBxAg) HBxAg is a common marker in the livers of carriers

Question 59

use of western blotting in cancer research

Answer 59

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

Question 60

Enzyme-Linked Immunosorbent Assay (ELISA) what is it used for

Answer 60

ELISAs are designed for detecting and quantifying substances such as ➢ peptides ➢ proteins ➢ antibodies ➢ hormones ➢ cytokines ➢ drugs of abuse and their metabolites

Question 61

name the 3 types of ELISA

Answer 61

1. indirect 2. sandwich 3. competitive

Question 62

explain Indrect ELISA

Answer 62

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

Question 63

applications of indirect ELISA explain the MOA of its application

Answer 63

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

Question 64

sandwich ELISA

Answer 64

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

Question 65

applications of sandwich ELISA

Answer 65

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

Question 66

competitive ELISA procedure

Answer 66

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

Question 67

applications of competitive ELISA

Answer 67

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