MOLECULAR DIAGNOSTICS

MOLECULAR DIAGNOSTICS
L. Duroux Slides assembled from diverse sources

Recommended reading list - textbooks
Molecular Diagnostics: A Training and Study Guide Gregory J. Tsongalis & William B. Coleman Amer. Assoc. for Clinical Chemistry , ISBN X Diagnostic Molecular Microbiology: Principles and Applications Thomas F. Smith, Fred C. Tenover, Thomas J. White & David H. Persing ASM Press, ISBN X

Journals Journal of Molecular Diagnostics Nature Biotechnology
Nature Biotechnology

Lecture Plan Introduction: Definitions, Problematics, Examples
Immunological Diagnostic Methods DNA Diagnostics Methods Bacterial Biosensors

1. INTRODUCTION

Molecular Diagnostics
The success of modern medicine depends on the detection of specific molecules e.g. Viruses Bacteria Fungi Parasites Proteins In water, plants, soil and humans.

Molecular Diagnostics are Transforming Medicine
Molecular diagnostics is >$3 billion market WW and growing at >20% annually Recurrence monitoring Drug selection Disease detection I’ve tried to make this section hard hitting and forward looking. Basically to make a clear statement that “we are here” “we are good” and “we are going to launch tests” Disease predisposition Key questions Pre-natal testing “Is the baby healthy? “ “What diseases is this patient at risk for?” “Do this patient have disease?” “What drugs should I prescribe?” “How the disease returned?” Need for Molecular tests

Characteristics of a Detection System
A good detection system should have 3 qualities: Sensitivity Specificity Simplicity Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules. Specificity: the test yields a positive result for the target molecule only. Simplicity: the test must be able to run efficiently and inexpensively on a routine basis.

MassARRAY Diagnostics Are Being Developed For Multiple Disease Areas
Genetic Testing High throughput testing for genetic disorders including SNPs, insertions, deletions Examples: Factor II, Factor V, CFTR Prenatal Diagnostics Non-invasive detection of fetal diseases Examples: Down syndrome, RhD, cystic fibrosis Progress is being made in all of these areas Each of these areas are commercially attractive In some cases, the MassARRAY platform is uniquely qualified for specific tests More tests will be added to the platform as these tests are rolled out Oncology Early diagnosis of cancer Example: circulating tumor DNA Transplantation Medicine Non-invasive, early detection of organ rejection Example: urine testing for kidney rejection Pathogen identification and early detection Examples: identification of multi drug resistant mycobacteria, early detection of drug-resistant viral strains, e.g. HIV, HBV, HCV Infectious Disease

Pre-natal Diagnostics
Example 1 Pre-natal Diagnostics

Non-invasive Pre-Natal Diagnostics: A Large, Untapped Market
130 million live births worldwide per year 8 million live births in US and Europe per year 6% of all babies are born with birth defects over 900 fetal genetic disorders Down syndrome is the most common chromosomal abnormality Although risk increases with age, 80% of Down births are in women <35 years old Even though limited to high risk mothers, amniocentesis is a $600 million market in US and $1.5 billion market worldwide Although market is large and there is unmet need, there are no accurate, non-invasive prenatal diagnostic tests (“NIPD”) available

Non-Invasive Prenatal Testing
Platform independent Mass spec, RT-PCR, others Covers all prenatal tests RhD – Commercialize homebrew in 1H07 Hemoglobinopathies Thalassemias – Asian market Cystic fibrosis Others With advanced pre-analytics Aneuploidies – Down Syndrome

Example 2 Genetic testing

Molecular Genetic Testing
Disease Genes Identified Molecular Tests Offered in North America Molecular Tests Offered In Ontario nlm.nih.gov

Types of Mutations Tested
Few recurrent mutations? Point mutations? Many unique mutations? Disease Whole gene? Some exons? Deletions & duplications? Other mutations? Also with point mutations?

Diagnosis of Batten Disease: Neuronal Ceroid Lipofuscinoses
Batten disease is a fatal, inherited disorder of the nervous system that begins in childhood. It is the buildup of lipopigments in the body's tissues. Lipopigments are made up of fats and proteins.

Primer Extension Test for NCL Mutations
Normal Mutant C451T An enzyme called palmitoyl-protein thioesterase has been shown to be insufficiently active in the infantile form of Batten disease. This condition is now referred to as NCL1.

Multiplexed Analysis of 9 NCL Mutations
C70G IVS C622T G284V C451T A364T 1.02del A223C Normal delAT NCL2: late infantile form, mutations of an acid protease. NCL3: juvenile form, gene not been identified

2- Immunological Diagnostics Methods

Applications of Immunoassays
Analysis of hormones, vitamins, metabolites, diagnostic markers Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, glucocorticoids, Therapeutic drug monitoring: Barbiturates, morphine, digoxin Diagnostic procedures for detecting infection HIV, Hepatitis A, B, etc…

Antigen-Antibody Interactions - a bimolecular association
involving various non-covalent interactions Is similar to an enzyme-substrate interactions, but not lead to an irreversible chemical alteration

Strength of Antigen-Antibody Interactions
Cross-Reactivity Precipitation Reactions Agglutination Reactions Radioimmunoassay Enzyme-Linked Immunosorbent Assay Western Blotting Immunoprecipitation Immunofluorescence Flow Cytometry and Fluorescence Alternatives to Antigen-Antibody Reactions Immunoelectron Microscopy

Strength of Ag-Ab Interactions
Antibody affinity is a quantitative measure of binding strength combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag Antibody avidity Incorporates affinity of multiple binding sites True strength of the Ab-Ag interaction within biological systems The interaction at one site will increase the possibility of reaction at a second site High avidity can compensate for low affinity ( secreted pentameric IgM has a higher avidity than IgG )

Forward & reverse rate constants ( k1 & k-1)
Strength of Ag-Ab Interactions Forward & reverse rate constants ( k1 & k-1) Association & dissociation constants ( Ka & Kd ) for 3 ligand-Ab interaction High affinity complexes have high Ka values Very stable complexes have very low values of Kd

Sensitivity of various immunoassays

CROSS-REACTIVITY Antibody elicited by one Ag can cross-react with unrelated Ag. occurs if two different Ags share identical or very similar epitope (i) Cowpox antigens in vaccinia virus are cross-reactive to smallpox antigens in variola virus (∵ share similar or identical epitope ) (ii) Rabies & JE vaccine >>> encephalitis ( 뇌염 ) (: rabbit brain antigen contaminated vs human brain Ag ) (iii) Streptococcus pyogenes infection >>> heart & Kidney damage following infection (: cell wall proteins called M antigens vs Myocardial & skeletal muscle proteins ). (iv) Original antigenic sin. - The existence of long-lived lymphocytes & crossreactivity - Vaccination with one strain of flu elicited Ab responses to another flu strain. (v) cross-reacting bacterial Ag vs glycoproteins on RBC)

2. Cross-Reactivity ABO blood types - The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestine bacteria. - ABO blood-group antigens have subtle differences in the terminal residues of the sugars on glyco-proteins in RBC. - Providing the basis for blood typing test in blood transfusion

Agglutination Reactions
-The original home pregnancy test kit employed hapten inhibition (agglutination inhibition) to determine the presence or absence of human chorionic gonadotropin (HCG) >>> The kits currently on the market use ELISA-based assays. -Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella).

ELISA Addition of a specific antibody (primary antibody) which will bind to the test molecule if it is present. Washing to remove unbound molecules. Addition of secondary antibody which will bind to the primary antibody. The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase. Wash to remove unbound antibody. Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result.

ELISA One of many variants of the technique http://www. immunospot

Advantages of ELISA Sensitive: nanogram levels or lower Reproducible
Minimal reagents Qualitative & Quantitative Qualitative  Eg HIV testing quantitative assays  Eg Ther. Drug Monitoring Greater scope : Wells can be coated with Antigens OR Antibodies Suitable for automation high speed NO radiation hazards

ELISA: Many variants to detect Ab (HIV) to detect Ag to detect Ag
Detection based on enzyme catalyzed reactions (alkaline ⓟ, horseradish peroxidase, & β-galactosidase) : safer & less costly.

6. ELISA -> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited. The ELISPOT assay, a modification of the ELISA assay to determine quantitatively the # of cells in a population that are producing specific Ab or cytokine.

Western blotting : separates the components according to
their molecular weight. : the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current. : probed with Ab & then radiolabeled or enzyme-linked 2nd Ab. : a position is visualized by means of an ELISA reaction.

Immunoprecipitation EM showing a cell with magnetic beads attached
Ag-Ab attached to a synthetic bead complex >>> 0 labeling Ag with radiolabeled leucine, cysteine, or methionine → A radiolabeled Ag-Ab complex → 0 → SDS•PAGE → autoradiography - Ag-Ab complex + 2nd Ab attatched to a synthetic bead or magnetic beads >>> 0 or magnet EM showing a cell with magnetic beads attached to its surface via antibodies. Immuno-precipitates can be collected using magnetic beads coupled to a secondary antibody.

Immunofluorescence Fluorochromes
Fluorescein (490→517nm) Rhodamine (515→546nm) Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein. mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

Immuno Electron Microscopy
electron-dense labels absorb electrons. An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies (Ab against class II MHC labeled with 30nm gold particles, & another Ab against class I MHC w/ 15nm gold particles. (The density of class I exceeds that of class II) Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc portion.

Alternatives to Ag-Ab Reactions
Ag-Ab-Ab*→Ag-IgG-A/G* or Ag-Ab-biotin-(a)vidin* ① Protein A (from staphylococcus) & protein G (from streptococcus) - bind to rhe Fc region of lgG molecules (ka～108) - used to detect lgG molecules in the Ag-Ab complexes - used to isolate lgG molecules in the affinity columns ② Avidin (from egg whites) & streptavidin (from streptomyces avidinii) conjugated with an enzyme, fluorochrome, radioactive label) - bind to biotin (a vitamin) with higher affinity (ka～1015) - Ab can be labeled with (ka～1018)

3. DNA Diagnostic Systems

Problematics & Solutions

Genetic testing in individuals and populations
Does THIS patient have ANY mutation in ANY gene that would explain his disease? Does THIS patient have ANY mutation in THIS gene that might cause his disease? Does THIS patient have a 3-bp deletion of Phe codon in CFTR gene? NOT POSSIBLE TO SAY NEED LOTS OF EFFORTS TO ANSWER THAT IS A RIGHT QUESTION !!!

The choice of material to test
DNA most common; tested by PCR Sometimes tested by Southern blotting RNA RT-PCR allow to test genes directly, without breaking them into exons. Allow to detect alternative spliced isoforms. Allow to test for unknown mutations faster than in DNA

How to obtain DNA specimen
Blood sample (most common for adult testing); Mouthwashes or buccal scrapes (non-invasive); Chorionic villus biopsy samples (fetal DNA); Hair, semen (criminology) One or two cells removed from 8-cell embryo (in vitro fertilisation) Archived pathological specimens (typing dead peoples, tumor samples in paraffin blocks); Paper cards with blood drops on them

Methods of mutation scanning (when we do not know where is our mutation)
Sequencing -- most direct method; Detecting mismatches or heteroduplex DNA molecules; Single-strand conformational analysis; Protein truncation test (PTT); Detecting of deletions; Detection of methylation

DNA Diagnostic Systems
DNA Diagnostic Systems include: DNA Hybridization DNA Sequencing PCR Restriction endonuclease analysis RAPD (random amplified polymorphic DNA) DNA fingerprinting

Hybridization methods

DNA Hybridization Bacterial and viral pathogens may be pathogenic because of the presence of specific genes or sets of genes. Genetic diseases often are due to mutations or absence of particular gene or genes. These genes (DNA) can be used as diagnostic tools. This involves using a DNA probe during DNA hybridization. What is a DNA probe? How does DNA hybridization work?

DNA Hybridization For DNA hybridization:
A probe is needed which will anneal to the target nucleic acid. Attach the target to a solid matrix e.g. membrane. Denaturation of both the probe and target. Add the denatured probe in a solution to the target. If there is sequence homology between the target and the probe, the probe will hybridize or anneal to the target. Detection of the hybridized probe e.g. by autoradiography, chemiluminsence or colorimetric.

DNA hybridization movie

Example: Detection of Malaria
Malaria is caused by the parasite Plasmodium falciparum. The parasite infects and destroys red blood cells. Symptoms include fever, rashes and damage to brain, kidney and other organs. Current treatment involves microscopic observations of blood smears, which is labour intensive. Other methods e.g ELISA does not differentiate between past and present infection. Why?

Detection of Malaria A DNA diagnostic system would only measure current infection. (Why?) The procedure involves: A genomic library of the parasite was screened with probes for parasitic DNA. The probes which hybridized strongly (highly repetitive DNA) were tested further. The probes were tested for their ability to hybridize to other Plasmodium species which do not cause malaria and to human DNA.

Theoretical Depiction of Malaria Infection
& Immune Response Microbe Assays Serology Assays ELISA P/N #pfu/ml 250 20 IgM IgG Malaria Neutralizing Ab 2 Viremia precedes clinical illness. There is some crossover period where people are clinically ill are viremic, and are IgM positive; but this is a rare event., The typical patient presents with clinical illness and is NAT negative, IgM positive. -14 to DAYS POST ONSET illness

Detection of Malaria Probes which hybridized to P. falciparum only could be used as a diagnostic tool. The probe was able to detect 10 pg of purified DNA or 1 ng of DNA in blood smear. Other DNA probes were developed for the following diseases: Salmonella typhi (food poisoning) E. coli (gastroenteritis) Trypanosoma cruzi (chagas’ disease)

TaqMan® Probes Unbound probe free in solution
Probe and primer bind target, FRET occurs Light Emission Donor dye (Reporter) Acceptor dye (Quencher) Light Energy transfer Taq Taq extends and hydrolyzes probe, donor dye free to emit fluorescence --> accumulation of signal Taq Light Emission Light

Taqman Probe design 20-30 bp in length, Tm 10°C higher than primers.
35-65% G/C; more Cs than G’s. Can try as high as 80% or as low as 20% if the region is particularly GC or AT rich. Avoid runs of 3+ of the same nucleotide, especially G’s. 5’ base  G. When the probe and primers anneal to the target, the 5’ end of probe should be 3 nucleotides from the 3’ end of the primer on same strand (max of 10-12). Test that primers and probe are not complementary to each other. (delta G free energy at 25C should be greater than -2)

Molecular Beacons Probe in preferred closed structure DNA template
Reporter dye Quencher Stem Loop Light Grd. St. Quenching Probe hybridized to DNA template Light Light Emission

Molecular Beacon design
Begin design as for a hydrolysis probe. Tm of probe region should be 7-10°C above target annealing temp. To the chosen sequence add a stem 5-7 bp in length, with similar Tm as the probe region. Test folding using appropriate software (mfold). Check that there is no complementarity between primers and probe. Tm of probe alone and probe + complement should be verified experimentally

Molecular Beacon Melting Curve
Properly designed Molecular Beacons can effectively discriminate between targets with a single bp mismatch. Beacon + Target Beacon + 1bp mismatched target Beacon alone

FISH Diagnosis Analyse chromosomes Sexing for X-linked disease
Chromosome abnormalities Age related aneuoploidy Fluorescent instiu hybridisationis used to analyse chromosomal change byPGD

Genetic Analysis Prenatal Diagnosis Preimplantation Genetic Diagnosis
Chorionic villus sampling (CVS) 10-14wks Amniocentesis (15-16wks) Fetal blood sampling (FBS) Preimplantation Genetic Diagnosis early embryos IVF selection for transfer to the uterus Genetic analysis usually takes the form of prenatal diagnosis. So chromosomes or DNA can be analysed from chorionic villus samples, amniotic fluid or in rare case by fetal blood sampling. These all take place after the prenancy is established. Preimplantation genetic diagnosis as the name suggests is carried on embryos at an early stage before they implant in the uterus. In order to be able to access embyos at this early stage they are created by in vitro fertilization.

Couple with a genetic disorder
Inherited disorders Couple with a genetic disorder Affected Children Other family members Options Reproductive roulette No more children Adoption Gamete donation Prenatal diagnosis / termination PGD If we consider a couple with a known genetic disorder – they may already have affected children or there may be other family members with the disease. They have a number of reproductive options. The may chose to take a chance and hope that their next child is free of disease or they may chose not to have any children or they may consider adoption. Gamete donation or prenatal diagnosis followed by termination of affected pregnancies are other way to try and ensure a disease free child. None of these choices are easy and PGD offers an alternative route for couple to try for genetically unaffected children

Cleavage Stage Biopsy I going to show you a short video clip of an embryo biopy/ Here we see a cleavsge stage embryo held in place by a pipette. First a small amount of acid solution is applied to the zona pellucida in order to thin it an make a small hole. Then using a wider bore biopsy pipette a sinlge blastomere is aspirated from the embryo – and this is used for the genetic analysis

Sexing Embryos for PDG: FISH analysis of interphase nuclei
Chromosome X Chromosome Y Chromosome 16 Here we can see the results of an embryo sexing for an x-linked disease. The left hand side appears to be normal female cells with two X and 2 chromosome 16 signals. The one on the right is a male cell. Normal Female Normal Male

Chromosome Abnormalities
Translocations Robertsonian Only 13,14,15,21,22 Reciprocal Insertions Inversions Ring Chromosomes

PGD of Chromosome Abnormalities:
Robertsonian Translocation Carrier 45,XY,der(13q;14q)(q10;q10) Chromosome 13 Chromosome 14 Normal for Chromosomes 13 & 14 Monosomy 14

Reciprocal Translocation 46,XX,t(5;9)(q32;p13)
Preimplantation Genetic Diagnosis of Chromosomal Imbalance for Reciprocal Translocation Carriers using Triple-colour FISH 5q32 9p13 der der Reciprocal Translocation 46,XX,t(5;9)(q32;p13)

Aneuploidy Screening Older women likely to produce abnormal oocytes
Leads to chromosomally abnormal embryos increase in miscarriage lower pregnancy rate Chromosomes commonly involved 13, 16, 18, 21, X and Y Used for older women with recurrent IVF failure recurrent miscarriage PDG for aneuploidy screening is a special type of analysis and is used in order to try and improve IVF pregnanacy rate. As women age they are are more likely to produce abnormal oocyte which results in chromosomally abnormal embryos. Some chromosomes are commonly involve and these chromosomes are examined by FISH I older women with recurrent IVF failure or miscariage in order to select chromosomally normal embryos

Chromosomes in human embryos
NORMAL All cells uniformly diploid ABNORMAL All cells uniformly abnormal eg trisomy 21 MOSAIC Two or more cell lines present often diploid with aneuploid or tetraploid cells CHAOTIC Different chromosome pattern in every cell Previous studies by or group on human embryos donated for research have shown that there are four types of embryo. Those that are uniformly normal, or uniformly abnormal. Some embryos show mosacism which can affect every cell.

Sequencing methods

Sequencing (cost – DKK 50,00 per run)
As sequencing becomes more and more cheap, it pushes other methods backward. For sequencing of genomic DNA, every exon is amplified separately (Typical sequencing run – 500bp; typical exon size – 145 bp)

Example of Diagnostic for Duchenne Muscular Dystrophy (DMD)
X-linked and affect mainly males an estimated 1 in 3500 boys worldwide DMD encodes a large structural protein: dystrophin strengthen muscle cells by anchoring elements of the internal cytoskeleton to the surface membrane Mutated dystrophin leads to ”implosion” of muscle cells

DMD Mutation Types 60 40 20 Deletion Duplication Point %

DNA Sequencing Normal Carrier

Minisequencing by primer extension
DNA polymerase + one of the four labeled dNTPs = sequencing of one nucleotide + HPLC analysis

Principle of Pyrosequencing
Step 1 A sequencing primer is hybridized to a single stranded, PCR amplified DNA template, and incubated with the enzymes: -- DNA polymerase, -- ATP sulfurylase, -- luciferase -- apyrase, and the substrates: -- adenosine 5´ phosphosulfate (APS) -- luciferin.

Step 2 The first of four dNTP is added to the reaction. DNA polymerase catalyzes the incorporation of the dNTP into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of an equimolar quantity of pyrophosphate (PPi)

Step 3 ATP sulfurylase PPi  ATP. ATP used in the conversion of luciferin to oxyluciferin (visible light proportional to ATP production). The light is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Light signal is proportional to the number of nucleotides incorporated.

Step 4 Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

Addition of dNTPs is performed one at a time. thio triphosphate (dATP-alphaS) is used as a substitute for the natural (dATP), as natural dATP is not good for luciferase

Problems arising in mutation scanning
Example: Duchenne muscular dystrophy Problems: Gene is large, 2,4 Mb, 79 exons Hard to find point mutation 2. High Frequency of new mutations (30% of cases); 3. First mutation carrier is often a mosaic (blood may be not a mutation carrier)

DNA Polymerase-based Diagnostics

Polymerase Chain Reaction
PCR uses 2 sequence specific oligonucleotide primers to amplify the target DNA. The presence of the appropriate amplified size fragment confirms the presence of the target. Specific primers are now available for the detection of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi.

Using PCR to Detect for HIV
RT-PCR (reverse transcriptase PCR). HIV has a ssRNA genome. Lyse plasma cells from the potentially infected person to release HIV RNA genome. The RNA is precipitated using isopropanol. Reverse transciptase is used to make a cDNA copy of the RNA of the virus. This cDNA is used as a template to make dsDNA.

RT-PCR Diagnosis of HIV

Using PCR to Detect for HIV
Specific primers are used to amplify a 156 bp portion of the HIV gag gene. Using standards the amount of PCR product can be used to determine the viral load. PCR can also be used as a prognostic tool to determine viral load. This method can also be used to determine the effectiveness antiviral therapy.

Detecting DMD Deletions by PCR
60 40 20 Deletion Duplication Point %

Deletions in DMD Gene : dystrophin
BMD Intermediate DMD 5' 3' 1 10 20 30 40 50 60 70 Exons

DMD Deletion Carrier Analysis
by PCR/ size analysis

DNA Fingerprinting (RFLP)
RFLP = Restriction Fragment Length Polymorphism Regular fingerprinting analyses phenotypic traits. DNA fingerprinting analyses genotypic traits. DNA fingerprinting (DNA typing) is used to characterize biological samples e.g. In legal proceedings to identify suspects and clear others. Paternity testing

Restriction fragment length polymorphism (RFLP)

Very simple; Not allow high-throughput detection; Even as many restriction enzymes are known, some mutation sites do not correspond to any Rare endonucleases are difficult to work with, and often of a poor quality

Diagnostic restriction site could be introduced artificially by purposedly mismatched PCR primer

Diagnosis of sickle cell anemia by RFLP.
Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6th aa of the  chain of hemoglobin. A (normal) glutamic acid and S (sickle) valine. In the homozygous state SS the red blood cells are irregularly shaped. The disease results in progressive anemia and damage to heart, lung, brain, joints and other organ systems. This occurs because the mutant hemoglobin is unable to carry enough oxygen to supply these systems.

Diagnosis of Sickle Cell Anemia
The single mutation in hemoglobin cause a change in the restriction pattern of the  globin gene abolishing a CvnI site. CvnI site CCTNAGG (N = any nt) Normal DNA sequence CCTGAGG (A) Mutant DNA sequence CCTGTGG (S) Two primers which flank the mutant region of the  globin gene is used during PCR to amplify this region of the gene. The PCR products is digested with CvnI and separated by agarose gel electrophoresis.

Detection of Sickle cell anemia by PCR/RFLP

Random Amplified Polymorphic DNA (RAPD)
Another method widely used in characterization of DNA is RAPD. RAPD is often used to show relatedness among DNA populations. In this procedure arbitrary (random) primers are used during PCR to produce a fingerprint of the DNA. A single primer is used which must anneal in 2 places on the DNA template and region between the primers will be amplified.

Random Amplified Polymorphic DNA (RAPD)
The primers (8-10nt) are likely to anneal in many places on the template DNA and will produce a variety of sizes of amplified products. Amplified products are separated by agarose gel electrophoresis and visualized. If the samples have similar genetic make up then the pattern of bands on the gel will be similar and vice versa. This procedure is widely used to differentiate between different cultivars/varieties of the same plant. Issues to consider when using this procedure include reproducibility, quality of DNA, and several primers may have to be used.

RAPD fingerprint

PCR/OLA Oligonucleotide Ligation Assay
Like sickle cell anemia many genetic diseases are caused by mutant genes. Many diseases are caused by a single nucleotide (nt) change in the wild type gene. A single nt change can be detected by PCR/OLA

PCR/OLA

Bacterial Biosensors

Bacterial Biosensors Bacterial sensors can be used to test for environmental pollutants. Bacteria with bioluminescent are good candidates for pollutant sensors. In the presence of pollutants the bioluminescent decreases. The structural genes (luxCDABD) encodes the enzyme for bioluminescent was cloned into the soil bacteria Pseudomonas fluorescens. The cells that luminescence to the greatest extent and grew as well as the wild type were tested as pollutant sensors.

Bacterial Biosensors To screen water samples for pollutants (metal or organic) a suspension of P. fluorescens was mixed with the solution to be tested. After a 15 min incubation the luminescence of the suspension was measured. When the solution contained low to moderate levels of pollutants the bioluminescence was inhibited. The procedure is rapid, simple, cheap and a good screen for pollutants.

Bacterial Biosensor

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