notes m. pharma sem ii
TRANSCRIPT
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OVERVIEW OF GENE PROBE METHODOLOGY
GENE PROBES
• Gene probes are single-stranded DNA or RNA.• Gene probes hybridize to a target DNA or RNA sequence.• Probe and target base sequences must be similar to each other but,
depending on conditions, do not necessarily have to be exactly identical,i.e. their complementarity is not always 100%.
• Gene probes must be labelled otherwise the hybridization cannot bedetected.
• Gene probes are used in various blotting and in situ techniques for thedetection of nucleic acid sequences. In medicine they can help in the
identification of microorganisms and the diagnosis of infectious, inheritedand other diseases.
PROBE LABELS
Radiolabels
Probe nucleic acid can be labelled using radioactive isotopes, e.g. 32P, 35S, 125I,3H.
Detection is by autoradiography or Geiger-Muller counters.
Radiolabelled probes used to be the most common type but are less popular
today because of safety considerations. However, radiolabelled probes are themost sensitive, e.g. 32P labelled probes can detect single-copy genes in only 0.5µg of DNA. High sensitivity means that low concentrations of probe-targethybrid can be detected.
Non-radioactive labels
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These are safer than radiolabels and do not require dedicated rooms,glassware and equipment or staff monitoring, etc . but they are not generally assensitive.
Some examples:
• Biotin This label can be detected using avidin or streptavidin which havehigh affinities for biotin.
• Enzymes The enzyme is attached to the probe and its presence usuallydetected by reaction with a substrate that changes colour. Used in thisway the enzyme is sometimes referred to as a "reporter group".Examples of enzymes used include alkaline phosphatase andhorseradish peroxidase.
• Chemiluminescence In this method chemiluminescent chemicals attachedto the probe are detected by their light emission using a luminometer.
Safety awards slide show
• Fluorescence Chemicals attached to probe fluoresce under UV light. Thistype of label is especially useful for the direct examination of microbiological or cytological specimens under the microscope - atechnique known as fluorescent in situ hybridization (FISH).
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• Antibodies An antigenic group is coupled to the probe and its presencedetected using specific antibodies. Also, monoclonal antibodies havebeen developed that will recognize DNA-RNA hybrids. The antibodiesthemselves have to be labelled, e.g . using an enzyme.
LABELLING METHODS
Whatever label is used, it has to be attached to, or incorporated into, the nucleic
acid probe.
Here are 5 methods of labelling probes (for more details of each click on theunderlined link):
1. Nick translation
2. Primer extension
3. RNA polymerase
4. End labelling
5. Direct labelling
Amplification of target sequence
To increase the sensitivity of a probe system the target sequence can beamplified (i.e. more copies made of it). This enables detection of very smallamounts of the target which may be undetectable without amplification.
Target DNA can be amplified using the polymerase chain reaction (PCR) or using ordinary DNA polymerase. Both methods use primers but PCR producesexponential amplification of the target whereas the second method producesarithmetical amplification and less target in a given time.
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BRINGING PROBE AND TARGET TOGETHER
Probe and target sequence hybridize with each other but how this is broughtabout can vary. There are 4 main formats:
1. Solid support
Target (usually) is bound to a solid support such as a microtitre tray or filter membrane. Probe is added in solution and binds to target (if present) on solidsupport. After washing to remove unbound probe, hybridization is detected onthe solid support using whatever method is appropriate for the probe label, e.g.f or a radiolabelled probe and a filter membrane autoradiography could be used;for an enzyme labelled probe and a microtitre tray the colour change of asubstrate could be measured using an ELISA plate reader.
2. In solution
Both the probe and the target are in solution. Because both are free to move,the chances of reaction are maximized and, therefore, this format is generallyfaster than others.
3. In situ
In this format probe solution is added to fixed tissues, sections or smears whichare then usually examined under the microscope. The probe label, e.g. afluorescent marker, produces a visible change in the specimen if the targetsequence is present and hybridization has occurred. However, the sensitivitymay be low if the amount of target nucleic acid present in the specimen is low.This can be used for the gene mapping of chromosomes, and for the detectionof microorganisms in specimens.
4. Southern & Northern blots
After size fractionation of nucleic acids by electrophoresis, they are transferredto a filter membrane which is then probed. The presence of target is confirmedby detection of probe on the filter membrane, e.g . radiolabelled probe can bedetected by autoradiography and the location of the target sequence in thebands in the original gel determined.
Southern blots are used for DNA analysis.
Northern blots blots are used for RNA analysis.
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Both these techniques are covered in Section 4 (Analysis of DNA).
STRINGENCY
This term can be defined as:
"The conditions of hybridization that increase the specificity of binding betweentwo single-stranded portions of nucleic acid."
(Stringency can be applied to any hybridization reaction, not just probe andtarget, but it is convenient to cover it here. The same principles apply, for example, to hybridization of a primer to a sequence prior to DNA replication.)
The degree and specificity of binding between two portions of nucleic acid
depends on hydrogen bonding between bases on opposite strands and isaffected by 4 external factors:
1. Temperature
2. pH
3. Salt concentration
4. Presence of denaturants
Nucleic acid hybrids can tolerate a certain proportion of mismatched base pairs(mismatches) and still form stable duplexes, i.e. remain double-stranded.
Normal pairings in DNA are A=T and G=C (with double and triple hydrogenbonds respectively).
Mismatches are: A/G, A/C, G/T and C/T which do not form hydrogen bonds.
The greater the proportion of mismatches, the greater the chances of the twomolecules separating (denaturing). However, under conditions of low stringency
two nucleic acid strands which are, perhaps, only 80% homologous may stillremain together. Under conditions of high stringency the same two strandswould separate. Therefore, altering the conditions of stringency can be used toassess the degree of homology or complementarity between two nucleic acidstrands.
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Stringency can also be regarded as measures of the degree of mismatch thatcan be tolerated, and the level of specificity of a probe (or primer) for its target.This is summarized in the table below:
LOW STRINGENCY HIGH STRINGENCY
TEMPERATURE LOW HIGH
pH NEUTRAL EXTREME
SALT CONCENTRATION HIGH LOW/ZERO
DENATURANT CONCENTRATION LOW/ZERO HIGH
TOLERANCE OF MISMATCHES HIGH LOW
SPECIFICITY LOW HIGH
Of the 4 external factors affecting hybridization, temperature is the easiest tocontrol and use to assess the specificity of a probe for its target.
High stringency is not always required as it increases specificity of probe for itstarget. If probe is not a perfect match for the target (perhaps because exactsequence of target is not known or because the target varies in differentsamples) it would be best to lower the conditions of stringency to ensure
hybridization.
Also, by varying stringency, it is possible to use one probe for different tasks,e.g. a probe for Campylobacter bacteria will hybridize and identify the speciesCampylobacter jejuni only when used under high stringency , but under lowstringency will identify any Campylobacter species. In this case, stringency canbe altered depending on what bacteria require identification.
Mid-point temperature (Tm)
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Tm is the temperature at which half (50%) of the hybridized nucleic acidmolecules in a sample denature, i.e. strand separation occurs. This is usedbecause in a population of nucleic acid molecules there will always be somevariation and also because of chance they do not all denature simultaneously ata particular temperature. Measuring the Tm gives an indication of thecomplementarity between probe and target (i.e. how closely their basesequences match). Tm also provides a measure of the specificity of a probe for its target - the higher the Tm the greater the specificity.
The graph below illustrates the measurement of the mid-point temperature froma graph of percentage hybridization against temperature. The temperature axishas no values because these will vary depending on the degree of complementarity between probe and target. If complementarity is high thegraph will be shifted to the right and Tm will be high. If complementarity is low,the graph will be shifted to the left and Tm will be lower.
The slope of the graph provides an indication of the homogeneity of thepopulation of hybridizing nucleic acids molecules, i.e. their uniformity. Thesteeper the graph, the more homogeneous the population.
SHORT v. LONG PROBES
Short, oligonucleotide probes may be only 14-40 bases in length. Long probesmay be 100's of bases in length. Under conditions of high stringency, and other optimal conditions, a probe is capable of detecting a one base pair (bp) changein nucleic acid.
Advantages of short probes (compared to long):
• more stable with time longer shelf life.• simpler to make.• hybridize at more rapid rates, e.g. short probes may have reaction time of
less than 30 minutes. (C.F. long probes may have reaction time of 4-16hours.)
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Disadvantages (compared to long):
• the amount of label that each probe molecule can carry is more limited byits shorter length.
• sensitivity may be less (for above reason).
The above problems can be overcome to some extent by: amplifying the target,thus allowing more probe molecules to hybridize and so increasing the signaland sensitivity; and/or amplifying the signal from the probe label (reporter molecule).
Long probes are more tolerant of mismatches than short probes but it is theproportion of mismatches that counts not the actual number. Therefore, a shortprobe with 2 mismatches is less likely to hybridize to a target than a long probewith 2 mismatches. Under standard conditions of use it would be expected that
short probes would be more specific for their target than long ones.
USES OF GENE PROBES
Probes hybridize to complementary DNA or RNA sequences and have severaluses:
1. Southern blots
Detection of gel-fractionated DNA molecules transferred to a membrane. Thisincludes restriction fragment length polymorphism (RFLP) analysis.
2. Northern blots
As above but used for RNA.
3. Dot blots
Detection of unfractionated nucleic acid immobilized on a membrane.
4. Colony and plaque blots
Detection of immobilized nucleic acid on a membrane that has been releasedfrom lysed bacteria or phages.
5. In situ hybridization
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Direct detection of nucleic acid in clinical specimens.
APPLICATIONS OF GENE PROBES
Gene probes have 3 basic applications in medicine:
1. Detection of specific nucleic acid sequences
Such sequences may be diagnostic of disease, e.g. the detection of a sequenceunique to a particular microorganism would demonstrate its presence in aspecimen and, perhaps, confirm an infectious disease. This is the principle of probes designed to detect and identify various infectious agents, includingbacteria, protozoa and viruses. Probes can be especially useful for detectingmicroorganisms that grow slowly (e.g. Mycobacterium tuberculosis) or which
cannot be cultured on artificial growth media (e.g. all viruses).
However, they are not usually capable of distinguishing between viable (live)
and non-viable (dead) cells, which is an important consideration with, for example, food poisoning organisms - many of which are not harmful unlessalive. Another problem is designing a probe to target a unique sequence so thatit will only detect the organism of interest. Sometimes an organism may show aunique biochemical characteristic and a probe can be designed to target thegene of the enzyme involved. But it is rarely that easy!
There is more discussion of nucleic-acid based methods for the detection andidentification of microorganisms in the PCR section.
2. Detection of changes to nucleic acid sequences
A change to the DNA sequence is a mutation, e.g . deletion, insertion,substitution. Changes in certain gene sequences can cause inherited diseasesand their detection by probes can be diagnostic. Unfortunately, with someinherited diseases more than one type of mutation can cause the disease. Inwhich case, a probe may have to be used under low stringency (to allow
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hybridization to a range of sequences) or several probes used (a "battery") toensure that the target is "hit".
Some examples:
• cystic fibrosis (due to a 3 bp deletion).
• muscular dystrophies (due to various intragenic deletions).•
phenylketonuria (due to various mutations).• apolipoprotein variants (some are due to a 1 bp mutation).• sickle cell anaemia (due to a 1 bp mutation).
• α1-antitrypsin deficiency (due to approx. 50 different variants).
3. Detection of tandem repeat sequences
Tandem repeat sequences are 30-50 bps in length. Their size and distributionare distinctive for an individual.
They can be detected using probes and PCR. They are the basis of so-called
"DNA fingerprinting"
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which was developed by Alec Jeffreys at the University of Leicester, UK
It is used in forensic science to confirm the identity of a suspect from specimensleft at the scene of a crime, e.g . any body fluid, skin, hair.
This technique can also be used for paternity tests, sibling confirmation (or exclusion) and tissue typing.
Q. Work out who is the criminal (or father!) from the following gel prints:
You could look up more details on this technique in one of the suggestedreferences.
1. Nick translation
This is the commonest method of labelling DNA probes but the term "nicktranslation" is confusing. It has nothing to do with the translation of mRNA intopolypeptide chains.
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Stages:
1. Nicks are introduced into a DNA duplex using DNAse I enzyme whichproduces random breaks in the strands.
2. This damage is then repaired by the addition of DNA polymerase I enzyme inthe presence of free labelled nucleotides.
3. Labelled nucleotides are incorporated into DNA molecule which thenbecomes labelled.
More label can be incorporated into the probe by allowing the reaction toproceed for a longer time.
2. Primer extension
Stages:
1. DNA of interest is denatured to give single-strands.
2. Random primer sequences are added (or sequences unique to a particular sequence of interest).
3. DNA polymerase is added together with labelled nucleotides.
4. Complementary DNA strands are synthesized starting from the primer sequences and incorporating the labelled nucleotides. There is partial filling inof the gaps between the primers.
5. DNA is then denatured to release the labelled probe molecules.
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3. RNA polymerase
RNA polymerase enzyme and labelled ribonucleotides are used to synthesizedRNA probes using DNA as a template.
DNA template + RNA polymerase + labelled ribonucleotides labelled RNAprobe
End labelling
Label is attached to either 3' or 5' end of linear DNA or RNA.
e.g. T4 polynucleotide kinase can be used to catalyze the transfer of labelledphosphate from a nucleoside donor to the 5' - hydroxyl group of a
polynucleotide, oligonucleotide or nucleoside.
Activity (and hence sensitivity) is generally less than with other labellingmethods because the amount of label is limited by the number of ends!!!
5. Direct labelling
Nucleic acids can be labelled directly by the addition of labelled materials thatcombine with them. The process is equivalent to "staining".
e.g. iodination of DNA using 125
I.
Proteome
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The proteome is the entire complement of proteins expressed by a genome, cell, tissue or
organism. More specifically, it is the set expressed proteins at a given time under defined
conditions. The term is a portmanteau of proteins and genome.
The term has been applied to several different types of biological systems. A cellular proteome is
the collection of proteins found in a particular cell type under a particular set of environmentalconditions such as exposure to hormone stimulation. It can also be useful to consider an organism's
complete proteome, which can be conceptualized as the complete set of proteins from all of thevarious cellular proteomes. This is very roughly the protein equivalent of the genome. The term
"proteome" has also been used to refer to the collection of proteins in certain sub-cellular
biological systems. For example, all of the proteins in a virus can be called a viral proteome.
The proteome is larger than the genome, especially in eukaryotes, in the sense that there are more
proteins than genes. This is due to alternative splicing of genes and post-translational modifications
like glycosylation or phosphorylation.
Moreover the proteome has at least two levels of complexity lacking in the genome. When thegenome is defined by the sequence of nucleotides, the proteome cannot be limited to the sum of the
sequences of the proteins present. Knowledge of the proteome requires knowledge of (1) the
structure of the proteins in the proteome and (2) the functional interaction between the proteins.
Proteomics, the study of the proteome, has largely been practiced through the separation of proteins by two dimensional gel electrophoresis. In the first dimension, the proteins are separated
by isoelectric focusing, which resolves proteins on the basis of charge. In the second dimension,
proteins are separated by molecular weight using SDS-PAGE. The gel is dyed with Coomassie Blue or silver to visualize the proteins. Spots on the gel are proteins that have migrated to specific
locations.
The mass spectrometer has augmented proteomics. Peptide mass fingerprinting identifies a protein
by cleaving it into short peptides and then deduces the protein's identity by matching the observed peptide masses against a sequence database. Tandem mass spectrometry, on the other hand, can get
sequence information from individual peptides by isolating them, colliding them with a non-
reactive gas, and then cataloguing the fragment ions produced.
History
The term was coined by Marc Wilkins in 1994 in the symposium: "2D Electrophoresis: from
protein maps to genomes" in Siena, Italy, and was subsequently published in 1995[1], which was
part of his PhD thesis. Wilkins used it to describe the entire complement of proteins expressed by agenome, cell, tissue or organism.
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Metabolome
Metabolome[1] refers to the complete set of small-molecule metabolites (such as metabolicintermediates, hormones and other signalling molecules, and secondary metabolites) to be found
within a biological sample, such as a single organism. The word was coined in analogy withtranscriptomics and proteomics; like the transcriptome and the proteome, the metabolome is
dynamic, changing from second to second. Although the metabolome can be defined readilyenough, it is not currently possible to analyse the entire range of metabolites by a single analytical
method (see metabolomics). In January 2007 scientists at the University of Alberta and the
University of Calgary finished a draft of the human metabolome. They have catalogued andcharacterized 2,500 metabolites, 1,200 drugs and 3,500 food components that can be found in the
human body.
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GENOMICS
Genomics is a discipline in genetics concerned with the study of the genomes of organisms. The
field includes efforts to determine the entire DNA sequence of organisms and fine-scale genetic
mapping. The field also includes studies of intragenomic phenomena such as heterosis, epistasis, pleiotropy and other interactions between loci and alleles within the genome. In contrast, the
investigation of the roles and functions of single genes is a primary focus of molecular biology or
genetics and is a common topic of modern medical and biological research. Research of singlegenes does not fall into the definition of genomics unless the aim of this genetic, pathway, and
functional information analysis is to elucidate its effect on, place in, and response to the entire
genome's networks.[1]
he first genomes to be sequenced were those of a virus and a mitochondrion, and were done byFred Sanger . His group established techniques of sequencing, genome mapping, data storage, and
bioinformatic analyses in the 1970-1980s. A major branch of genomics is still concerned with
sequencing the genomes of various organisms, but the knowledge of full genomes has created the possibility for the field of functional genomics, mainly concerned with patterns of gene expression
during various conditions. The most important tools here are microarrays and bioinformatics.
Study of the full set of proteins in a cell type or tissue, and the changes during various conditions,is called proteomics. A related concept is materiomics, which is defined as the study of the
material properties of biological materials (e.g. hierarchical protein structures and materials,
mineralized biological tissues, etc.) and their effect on the macroscopic function and failure in their
biological context, linking processes, structure and properties at multiple scales through a materialsscience approach. The actual term 'genomics' is thought to have been coined by Dr. Tom Roderick,
a geneticist at the Jackson Laboratory (Bar Harbor, ME) over beer at a meeting held in Maryland
on the mapping of the human genome in 1986. The Genomic Science Program (formerly Genomesto Life) uses microbial and plants.
In 1972, Walter Fiers and his team at the Laboratory of Molecular Biology of the University of
Ghent (Ghent, Belgium) were the first to determine the sequence of a gene: the gene for
Bacteriophage MS2 coat protein.[3] In 1976, the team determined the complete nucleotide-sequenceof bacteriophage MS2-RNA.[4] The first DNA-based genome to be sequenced in its entirety was
that of bacteriophage Φ-X174; (5,368 bp), sequenced by Frederick Sanger in 1977.[5]
The first free-living organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in
1995[6], and since then genomes are being sequenced at a rapid pace. As of October 2011, thecomplete sequences are available for: 2719 viruses,[7] 1115 archaea and bacteria, and 36
eukaryotes, of which about half are fungi. [8]
Most of the bacteria whose genomes have been completely sequenced are problematic disease-
causing agents, such as Haemophilus influenzae.[9] Of the other sequenced species, most werechosen because they were well-studied model organisms or promised to become good models.
Yeast (Saccharomyces cerevisiae) has long been an important model organism for the eukaryotic
cell, while the fruit fly Drosophila melanogaster has been a very important tool (notably in early
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Protein engineeringProtein engineering is the process of developing useful or valuable proteins. It is a young
discipline, with much research taking place into the understanding of protein folding andrecognition for protein design principles.
There are two general strategies for protein engineering, rational design and directed evolution.
These techniques are not mutually exclusive; researchers will often apply both. In the future, more
detailed knowledge of protein structure and function, as well as advancements in high-throughput technology, may greatly expand the capabilities of protein engineering. Eventually, even unnatural
amino acids may be incorporated thanks to a new method that allows the inclusion of novel amino
acids in the genetic code.
The number of possible amino acid sequences is enormous, but only a subset of them will fold reliably and quickly to a single native state. Protein design involves identifying novel sequences
within this subset, in particular those with a physiologically active native state. Physically, the
native state of a protein is the conformational free energy minimum for the chain. Therefore protein design is the search for sequences which have the chosen structure as a free energy
minimum. In a sense it is the reverse of structure prediction: in design, a tertiary structure is
specified, and a sequence is identified which will fold to it. Hence it is also referred to as inverse folding .
Prion diseases like mad-cow disease illustrate how important it is that designer proteins possessonly one stable conformation. In mad-cow disease, there exists a healthy protein with a fatal
weakness: there is another conformation that it can "comfortably" take; the abnormally foldedshape has very little free energy and is therefore very stable. For reasons that are not yet fully
understood, this mis-folded prion protein can catalyze other proteins of its type to also adopt the
mis-folded shape, causing a disease-generating cascade of previously functional proteins to quicklymis-fold. They lose the ability to perform their intended function in the new conformation, and
have a tendency to form aggregates called plaques. The buildup of these aggregates in the brain
leads to progressive neuronal death, and eventually death of the entire organism. It is therefore
easy to see the importance both that a designer protein have only one possible stable tertiarystructure, and that researchers exercise extreme diligence to ensure that this remains the case in all
environments – especially in vivo.
Examples of designed proteins
The early 21st century saw the creation of small proteins with real biological functions including
chiroselective catalysis,[1] ion detection,[2] and antiviral behaviour.[3] Using computational methods,
a protein with a novel fold (Top7) was designed in 2003,[4] as well as sensors for unnatural
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molecules.[5] Recent computational redesign was capable of experimentally switching the cofactor
specificity of Candida boidinii xylose reductase from NADPH to NADH.[6]
On the other hand, it is widely believed that not all possible protein structures are designable,which means that there are compact configurations of the chain which no sequences can fold to. In
particular, conformations which are poor in secondary structures are unlikely to be designable. Thedesignability of given structures is still poorly understood.
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DNA Microarrays - A technology that is reshaping molecular
biology
It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism
function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in
molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very
limited and the "whole picture" of gene function is hard to obtain. In the past several years, a new technology, called
DNA microarray, has attracted tremendous interests among biologists. This technology promises to monitor the
whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously.
Terminologies that have been used in the literature to describe this technology include, but not limited to: biochip,
DNA chip, DNA microarray, and gene array. Affymetrix, Inc. owns a registered trademark, GeneChip®, which refers
to its high density, oligonucleotide-based DNA arrays. However, in some articles appeared in professional journals, popular magazines, and the WWW the term "gene chip(s)" has been used as a general terminology that refers to the
microarray technology. Affymetrix strongly opposes such usage of the term "gene chip(s)". More recently, I prefer the term "genome chip", indicating that this technology is meant to monitor the whole genome on a single chip.
GenomeChip would also include the increasingly important and feasible protein chip technology.
Base-pairing (i.e., A-T and G-C for DNA; A-U and G-C for RNA) or hybridization is the underlining principle of
DNA microarray.
An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA
samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by
hand or make use of robotics to deposit the sample. In general, arrays are described as macroarrays or microarrays,
the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically
less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require
specialized robotics and imaging equipment that generally are not commercially available as a complete system.
DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon
substrates, for which probes* with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide
researchers information on thousands of genes simultaneously - a dramatic increase in throughput. (*Note: In the
literature there exist at least two confusing nomenclature systems for referring to hybridization partners. Both use
common terms: " probes" and "targets". According to the nomenclature recommended by B. Phimister of Nature
Genetics, a "probe" is the tethered nucleic acid with known sequence, whereas a "target" is the free nucleic acid
sample whose identity/abundance is being detected. This site follows that recommendation. See Nature Genetics
volume 21 supplement pp 1 - 60, 1999, which is freely accessable.
There are two major application forms for the DNA microarray technology: 1) Identification of sequence (gene / gene mutation); and 2) Determination of expression level (abundance) of genes.
There are two variants* of the DNA microarray technology, in terms of the property of arrayed DNA sequence with
known identity:
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Format I : probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass using robot
spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called
DNA microarray, is widely considered as developed at Stanford University. A recent article by R. Ekins
and F.W. Chu (Microarrays: their origins and applications. Trends in Biotechnology, 1999, 17, 217-218)
seems to provide some generally forgotten facts.
Format II : an array of oligonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The
array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary
sequences are determined. This method, "historically" called DNA chips, was developed at Affymetrix,
Inc. , which sells its photolithographically fabricated products under the GeneChip® trademark. Many
companies are manufacturing oligonucleotide based chips using alternative in-situ synthesis or
depositioning technologies.
In the preparation of this Web site, "DNA microarray(s)" and "DNA chip(s)" are used interchangeably. But
viewers should aware this technical difference.
* In addition, microfluidics-based chip or laboratory-on-a-chip systems are also listed in this Web site.
The microarray (DNA chip) technology is having a significant impact on genomics study. Many fields, including drug discovery and toxicological research, will certainly benefit from the use of DNA microarray technology. View an example of the microarray image (38K).
For a very well-written introduction on the steps involved in a microarray experiment, visit Jeremy Buhler's
Anatomy of a Comparative Gene Expression Study
An excellent collection of Genomics Glossaries (including a Microarrays Glossary) is being maintained by Mary
Chitty of Cambridge Healthtech Institute.
Design of a DNA Microarray System
There are several steps in the design and implementation of a DNA microarray experiment. Many strategies have been investigated at each of these steps. 1) DNA types; 2) Chip fabrication; 3) Sample preparation; 4) Assay; 5)
Readout; and 6) Software (informatics)
Table 1. Steps in the design and implementation of a DNA microarray experiment
1) Probe
(cDNA/oligo
with known
identity)
2) Chip fabrication
(Putting probes on
the chip)
3) Target
(fluorecently
labeled sample)
4) Assay 5) Readout 6) Informatics
Small oligos,
cDNAs, chromosome,
... (whole
Photolithography,
pipette, drop-touch,
piezoelectric (ink-
jet), electric, ...
RNA,
(mRNA==>)
cDNA
Hybridization,
long, short, ligase,
base addition,
electric, MS,
electrophoresis,
fluocytometry,PCR-DIRECT,
TaqMan, ...
Fluorescence,
probeless
(conductance, MS,
electrophoresis),
electronic, ...
Robotics control,
Image processing,
DBMS, WWW,
bioinformatics,
data mining and
visualization
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organism on a chip?)
There are so many options and combinations, as can been seen from the number of companies involved in this business. It seems too early to judge who will be the winner(s) in this game. The forecast is further complicated by
recent fights among companies on intellectual property issues.
Applications of DNA Microarray Technology
Gene discovery
(Many, many applications, to be listed)
Disease diagnosis
(Many, many applications, to be listed).
Many "microfluidics" devices (Chemical & Engineering News, February 22, 1999, 77(8):27-36; password
required) fall in this category. Although they are not the "traditional" gene chip or microarray, I decided to list related links at this site because of their close connection and integration to the gene chip (microarray)
technology.
Drug discovery: Pharmacogenomics
Why some drugs work better in some patients than in others? And why some drugs may even be highly
toxic to certain patients? My favorite definition (modified): Pharmacogenomics is the hybridization of
functional genomics and molecular pharmacology. The goal of pharmacogenomics is to find correlations
between therapeutic responses to drugs and the genetic profiles of patients.
Toxicological research: Toxicogenomics
Have you seen anybody using this terminology? Now let's try to give it a definition: Toxicogenomics is the
hybridization of functional genomics and molecular toxicology. The goal of toxicogenomics is to find
correlations between toxic responses to toxicants and changes in the genetic profiles of the objects exposed
to such toxicants. First Preclinical Toxicity Application (Toxicology EXPRESS™ database using Gene
Logic's Flow-thru Chip™ technology) between Wyeth-Ayerst Research and Gene Logic
An interesting article: Nuwaysir, E.F., Bittner, M., Trent, J., Barrett, J.C., and Afshari, C.A. Microarray and
Toxicology: The Advent of Toxicogenomics. Molecular Carcinogenesis, 24:153-159(1999).
NIEHS sponsored a meeting on the application of DNA microarray in toxicology (EHP 1999). NIEHS established the National Center for Toxicogenomics ( NCT) in June 2000.
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Protein microarrays
Protein microarrays are tools that can be used in many different areas of research, including basic
and translational research. Protein arrays can take on many different formats and can be used to domore than simple expression profiling of samples.
Recent publications have demonstrated that protein microarrays can be used to phenotype
leukemia cells, identify novel protein-protein interactions, screen entire proteomes for new proteins, and profile hundreds of patient samples simultaneously. Whatman has led the way in
protein array technology starting with the development of the FAST Slide: the premier protein
arraying surface.
Proteomic Arrays
Proteomic arrays are typically high-density arrays (> 1000 elements/array) that are used to identify
novel proteins or protein-protein interactions. The library that is arrayed can come from many possible sources, including expression libraries, and can contain known, as well as unknown,elements. The sample to probe the array can come from virtually any source.
To detect proteins that are bound to the array, the samples must be labeled directly with a
fluorophore or a hapten. Alternatively, in some applications antibodies can be used to detect
binding events. One common use is for antibody screening.
Microspot ELISA and Antibody Arrays
Microspot ELISA and antibody arrays are used for quantitative profiling of protein expression in
cell cultures or clinical specimens. Typically these arrays are low-density (9 to 100
elements/array). In these arrays, known antibodies are arrayed and used to capture antigens fromunknown samples. To detect antigen that is bound to the array, the antigen either needs to be
labeled directly with a fluorophore, or a second binder/antibody can be used. The latter option
creates a sandwich assay similar to a traditional ELISA, only in a microspot format. The WhatmanFAST Quant Cytokine Quantification Kits offer an example of a microspot sandwich assay.
Single-Capture Antibody Arrays
Single-capture antibody arrays consist of multiple, known antibodies arrayed to a solid surface andused to profile the presence of specific antigens from a pooled sample, usually consisting of both a
normal and disease-present sample. A single capture antibody array utilizes a direct or hapten
labeling system, which does not require a matched antibody. Single-capture antibody arrays offer a
qualitative profiling tool to detect binding events. The Whatman Serum Biomarker Chip offers anexample of a single-capture antibody array.
Antigen Arrays or Reverse Arrays
One application of antigen arrays is to interrogate research or clinical samples for the presence of
auto-antibodies. Normally, a low-density array is probed with serum or plasma samples. TheWhatman CombiChip Autoimmune Kit offers an example of an antigen array. Reverse arrays are
used to profile dozens or hundreds of samples (research or clinical) for the presence of a small
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number of antigens (1 to 3). Cell lysates, material from laser capture microdissection or serum
samples are arrayed. This creates an array of ‘unknowns’ that can be probed with a small number
of antibodies. Visualization can be performed with a detection or ‘top’ antibody linked to afluorophore or color detection reagent.
Microarray WesternsAn alternative strategy for protein microarrays is to array samples containing multiple proteins on
the FAST Slide and probe with a labeled antibody or set of antibodies. The advantage of the microformat is that extracts from various treatments and time points can be arrayed on the same slide.
Once arrayed, the levels of multiple proteins can be measured and compared simultaneously.
Protein Binder ArraysProtein arrays can be used to identify novel protein binding motifs or protein-protein interactions.
Engineered or synthetic proteins, or peptides with various binding motifs are arrayed, and the array
is probed with complex protein samples. Detection with a known antibody allows the researcher to
identify previously unknown binding events.