Unit 7: Techniques and diagnostics

Western Blotting in cancer research

"Cell Signaling Technology" sells an 'Oncogene and Tumour Suppressor Sampler Kit' containing primary antibodies against eight proteins including BRCA1, HER2, p53, phospho-estrogen recepter

PCR - Medical applications (6)

1. Genetic testing 2. Pre-natal testing 3. Pre-implantation genetic diagosis 4. Tissue typing 5. Diagnosis of Infectious Disease e.g. Human Immunodeficiency Virus or Human Papilloma Virus or Hepatitis 6. Many forms of cancer involve alterations to genes

PCR - Disadvantages (4)

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 (5kb have been done) 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

Process of SDS PAGE (2)

1. 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 2. The technique involves the 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.

Protein microarray 3 steps

1. 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. 2. Probe molecules, typically labeled with a fluorescent dye, are added to the array 3. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner

Process of western blotting (3)

1. The gel is blotted onto a positively charged membrane that traps the charged proteins and immobilizes them on the surface of the membrane (by applying an electric field to the gel to drive the proteins horizontally out of the gel onto the blotting membrane) 2. The blot can then be probed with antibodies directed against the protein (antigen) (Ag) of interest 3. The Ag-Ab complexes that form on the band containing the protein recognized by the antibody can be visualized in a variety of ways

indirect ELISA process (4)

1. The simplest format involves coating of antigen onto microtitre plates, followed by incubation with a specific antibody. 2. The binding antibody or an appropriate secondary antibody is conjugated to an enzyme that typically catalyzes formation of a colored product 3. enzymes ABTS or TMB produce changes in color which act as signal 4. Color formation is monitored spectophotometrically and related to concentration of antigen by calibration to a standard curve

PCR - Advantages (3)

1. Time taken to amplify sufficient amounts of the target sequence: a single molecule of the target sequence can be amplified to 10^9 copies in 1.5-6 hours 2. Sensitivity: A single copy of the target DNA sequence can be amplified rapidly to usable concentrations 3. 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

RT-PCR - Applications (2)

1. the diagnosis of genetic diseases 2. the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression

how much would ngst be

1000

Microarrays are manufactured by high-speed robotics which can put thousands of samples on a glass slide with an area of

1cm^2

how much is sangers per genome

20 million

PCR is usually carried out in ________________ volumes in PCR tubes.

20-50 microL

Generally, serum antibodies to HIV can be detected by indirect ELISA within

6 weeks of infection

RT-PCR is

A variation on PCR, where mRNA or total RNA is purified from the sample and converted to complementary DNA (cDNA) by the enzyme reverse transcriptase then amplified by PCR

Sanger 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

Western blotting needed because

Antibodies (Ab) are relatively large molecules and cannot penetrate the gel matrix

MammaPrint is an example of

Biomarkers of stage 1 or 2 node-negative breast cancer subtypes were identified using gene expression patterns in microarrays - using hierarchical clustering of gene expression

qPCR is used in tandem with RT-PCR to diagnosis what?

COVID19

2 other methods of DNA sequencing

Chemical Degradation Method and Pyrosequencing

dye used in SDS PAGE

Comassie Blue

What catalyzes strand synthesis rxn in sangers

DNA Polymerase

A basic PCR set up requires several components and reagents including (5)

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

ELISA is

Enzyme-linked Immunosorbent Assay

What molecule is qPCR done with?

Fluorescent label

competitive vs indirect elisa

In competitive ELISA, the higher the concentration of antigen in the original sample, the lower the absorbance

6 steps of microarray for drug testing

In step 1, a microarray is acquired that has a single-stranded DNA representing thousands of different genes In step 2, different populations of liver cells are collected, one treated with the potential drug and the other untreated, mRNA isolated In 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 In step 4, the labelled cDNAs are added to the DNA chips, the cDNAs bind to the chip if they find their complementary sequences in the single-stranded DNAs loaded onto the chip In step 5, the chip is scanned and a computer analyses the fluorescence: green dot means rna produced in untreat red dot means rna produced in treat yellow dot means produced in both blank dot means neither Step 6 : To answer the question of whether the potential drug is toxic to liver cell, compare to previous tests (controls) using toxic and nontoxic drugs

_______ can be used to detect the bcr-abl oncogene for CML and determine which variant of the gene is present

PCR

DNA Diagnostics: 5 methods

Polymerase Chain Reaction (PCR) Reverse Transcriptase PCR (RT-PCR) Real time PCR DNA Sequencing & Next Generation DNA Sequencing DNA Microarrays

RT-PCR for stage 1 or 2 node-negative breast cancer

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

Quantitative PCR aka

Real Time PCR

RT-PCR full

Reverse Transcriptase PCR

4 types of NGSTs

Roche 454 Sequencing Applied Biosystems/ SOLiD Illumina Genome Analyzer Helicos

3 steps of PCR and temps

Step 1: Denaturation of the template DNA at 94oC Step 2: Annealing of the single-stranded primers at 55 - 65oC. Step 3: Extension of the annealed primers by addition of nucleotides by base pairing to the template DNA at 72oC.

5 steps 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 PlateTM. Step 5: The DNA fragments on each bead are sequenced by pyrosequencing using a 454 Sequencer

In mammaprint, large gene sets (70 genes) were able to identify (5)

Subtypes of breast cancer including: basal-like (often seen in carriers of BRCA1 mutations), HER2-overexpressing subtype, two types of luminal cells normal tissue-like subgroup

PCR is named after

Taq DNA Polymerase from Thermus aquaticus

Western Blotting - Applications (4)

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. Creutzfeld- Jakob 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

transcriptome is

The genes that are transcribed at any particular time are known as the

how to amplify sensitivity of western blotting

a chemiluminescent compound with suitable enhancing agents is used to produce light at the antigen site

Each ddNTPS is labeled with

a different fluorescent marker

Agarose is a polysaccharide which acts as

a molecular sieve

A microarray could be used to determine if _________________ would be harmful to the liver

a new anti-cancer drug

PCR products are visualized by

agarose gel electrophoresis

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

DNA Microarray (DNA chip or gene chip) has been used to analyse

an entire genome in one experiment

example of use of protein aray

antibodies to known diseases can be bound to the microarray

ELISA is the most common and widely used

antibody application

competitive ELISA procedure? (4)

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

limitation of antibodies protein array

availability of antibodies

Once incorporated, a dideoxynucleotide

blocks further strand elongation because it lacks the 3'-hydroxyl group needed to form the connection with the next nucleotide

4 deoxyribonucleotides triphosphates (dNTPS) used in sangers

dATP, dTTP, dCTP and dGTP

are there more dNTPS or ddNTPS in dna

dNTPS

4 ddNTPS used in sangers

ddATP, ddTTP, ddCTP and ddGTP not as many as DNTPS

Polymerase enzyme does not discriminate between

deoxy- and dideoxynucleotides

the most generally used detection procedures to visualize western blotting

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

Gel contains _________ dye (or SYBR Green) to allow DNA to be seen under ultraviolet light

ethidium bromide

ddATP results in what

generated a family of 'A' terminated molecules

Sandwich ELISA clinical uses (4)

hCG pregnancy test mucins in pancreatic cancer CD44 in bladder cancer FDP via onko-sure test in colorectal, breast, lung, and ovarian cancer

western blot aka

immunoblot

With each of PCR cycle the number of DNA strands whose 5' and 3' ends are defined by the ends of the primers

increases exponentially

3 types of ELISA

indirect, sandwich, competitive

As PCR progresses, the DNA thus generated is

itself used as a template for replication

Sandwich ELISA is used to measure

large antigens

indirect ELISA used to

measure antibodies e.g. antibodies against HIV or an anti-cancer antibody cofirmatory tests for western blot and pcr

As a result, the desired DNA is preferentially replicated until after 20-30 cycles, it makes up

most of the DNA in the tube

DNA is ___________ charged and is attracted to the ___________ anode

negatively / positive

use of ddNTPS results in

new set of 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

NGST used in understanding and treatment of what type of cancer

pancreatic

ELISAs are designed for detecting and quantifying substances such as (6.5)

peptides proteins antibodies hormones cytokines drugs of abuse and their metabolites

The principle behind the microarray is the (3)

placement of specific nucleotide sequences in an ordered array 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

washing done repeatedly in

sandwich ELISA

Microarrays can be used in diagnosis via

scan cells from cancer patients and correlate microarray patterns with prognosis

Sanger sequencing requires ____________ DNA

single-stranded

Competitive ELISA is used to measure (1+3)

small antigens e.g. steroids (oestradiol 17β), drugs, peptides (insulin)

SDS-PAGE stands for

sodium dodecyl sulfate polyacrylamide gel electrophoresis

dideoxynucleotide triphosphates (ddNTPS) are

terminating nucleotides

Quantitative PCR monitors

the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR

Sandwich ELISA different from indirect ELISA because

the antibody (rather than antigen) is immobilized on a microtitre well, antigen added, then second antibody added before color substrate

Protein microarrays main advantage

the fact that large numbers of proteins can be tracked in parallel

SDS PAGE used to determine

the relative molecular mass of a protein and characterize the proteins found in a sample e.g. blood serum

PCR: In the _______ cycle, two double-stranded DNA molecules that exactly match the target sequence are produced

third

Competitive ELISA - Cancer Applications (3)

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)

A protein microarray (or protein chip) is a high-throughput method used to (3)

track the interactions and activities of proteins, and to determine their function, and determining function on a large scale

autoradiography is

used in western blotting, 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

DNA Microarray can be used to study gene expression and transcription rates of the genome in

vivo