Unit 7 - Techniques and Diagnostics Flashcards Preview

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Flashcards in Unit 7 - Techniques and Diagnostics Deck (67)
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1
Q

DNA diagnostic tests

A

PCR

Reverse Transcriptase PCR (RT-PCR)

real time PCR
DNA sequencing and Next Generation DNA sequencing

DNA microarrays

2
Q

what is PCR named after

A

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

3
Q

MOA of PCR

A

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

4
Q

what is needed for PCR

A

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

5
Q

thermal cycling

A

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

6
Q

3 steps of PCR procedure

A
  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
7
Q
A
8
Q

1st cycle

A

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

9
Q

2nd cycle

A

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

 

10
Q

3rd cycle

A

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

11
Q

what changes with each cycle

A

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)

12
Q

what is Agarose Gel Electrophoresis

how does it work

A

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

13
Q

advantages of PCR

A

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

14
Q

disadvantages of PCR

A

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

15
Q

5 medical applications of PCR

A

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

16
Q

how is PCR used in cancer diagnostics

A

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

17
Q

PCR and the bcr-abl oncogene

how is bcr-abl formed

A

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)

18
Q

applications of RT-PCR

A

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)

19
Q
A
20
Q

Oncotype-DX

A

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

21
Q

MOA of real time/quantitative PCR

A

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

22
Q

uses for Chain Termination (Sanger) DNA Sequencing

A

genome projects - easily automated

(other methods include Chemical Degradation Method and Pyrosequencing)

23
Q

steps in the Chain Termination (Sanger) DNA Sequencing

 

A

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

24
Q

main events of Chain Termination (Sanger) DNA Sequencing

A

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

25
Q

what happens during electrophoresis in Sanger DNA Sequencing

how is the information interpreted

A

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

26
Q

Next Generation Sequencing Technologies (NGST) types

A

Roche 454 Sequencing

Applied Biosystems/ SOLiD

Illumina Genome Analyzer

Helicos

27
Q

MOA of Roche - 454 Sequencing

A

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

28
Q

Pyrosequencing (put in steps)

A
29
Q

how has NGST helped in clinical practice

A

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

30
Q

interaction between genes and their product

A

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

31
Q

DNA microarray (DNA chip or gene chip) analyses

A

an entire genome in one experiment

32
Q

what can the DNA microarray be used to study

A

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

33
Q

transcriptome

A

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

34
Q

principle behind the microarray

A

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

35
Q

what could a microarray be used to determine

A

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

36
Q

microarray step 1

A

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

37
Q

microarray step 2

A

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

38
Q

microarray step 3

A

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

39
Q

microarray step 4

A

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

40
Q

microarray step 5

A

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

41
Q

microarray step 6

A

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

42
Q

overview of microarray

A
43
Q

how can microarrays be used in diagnosis

A

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

44
Q

MammaPrint

A

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

45
Q

what does whole genome expression profiling allow for

A

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

46
Q

protein microarray

what is its advantage

 

A

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

 

47
Q

how does protein microarray work

A

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

48
Q

advantages of protein microarray

uses

A

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

49
Q

scan to detect anthrax

A
50
Q

what is protein microarray limited by

A

availability of antibodies

51
Q

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE)

uses

 

 

 

A

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

52
Q

overview of MOA - SDS PAGE

A

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

53
Q

Western Blotting - why is it needed

A

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

54
Q
A
55
Q

MOA of Western Blotting

A

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

56
Q

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

A

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

57
Q

applications of Western Blotting

A

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

58
Q

how does Hepatitis B infection work

how can it be detected

A

the genome of hepatitis B virus (HBV) encodes the so-called "X" protein (HBxAg)

HBxAg is a common marker in the livers of carriers

 

59
Q

use of western blotting in cancer research

A

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

60
Q

Enzyme-Linked Immunosorbent Assay (ELISA)

what is it used for

A

ELISAs are designed for detecting and quantifying substances such as

➢ peptides

➢ proteins

➢ antibodies

➢ hormones

➢ cytokines

➢ drugs of abuse and their metabolites

61
Q

name the 3 types of ELISA

A
  1. indirect
  2. sandwich
  3. competitive
62
Q

explain Indrect ELISA

A

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

63
Q

applications of indirect ELISA

explain the MOA of its application

A

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

64
Q

sandwich ELISA

A

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

65
Q

applications of sandwich ELISA

A

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

66
Q

competitive ELISA procedure

A

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

67
Q

applications of competitive ELISA

A

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