Microarrayswith an emphasis on DNA microarrays

BE 4332 Final ProjectNatalie Derise

Formal Definition

A microarray is a hybridization-based technique that allows simultaneous analysis of thousands of samples of biological material on a solid substrate.

Common Types of Microarrays

Type of array

General function

Protein Protein expression and interactions profiling

TissueCompare histologic sections from unique tissues or tumors; tissues from multiple patients on same

slide

CellularReverse transfection and PMHC; study cell

responses

AntibodyDetects antigens and protein expression; many

diagnostic applications

DNA Measure gene activity and genotyping Additional types of assays: • Chemical compound microarrays• Carbohydrate microarrays• Phenotype micro arrays• Many more specific types under

development

DNA Microarrays

• A DNA microarray consists of pre-designed synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix.

• DNA microarrays rely on the hybridization between cDNA that is reverse transcribed from a biological sample to the pre-designed probes on the array.

Common Terms• Array: refers to the glass, plastic, or silicon slide

that the DNA probes will be spotted or built on.• Target DNA/RNA*: the nucleic acid (cDNA or

cRNA) sample that is being identified and/ or measured.

• Probe: short sections of oligonucleotides that are attached to the array and hybridize with the target cDNA or cRNA

• Hybridization: the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing.

*RNA microarrays are very similar to DNA microarrays, but involve RNA probes hybridizing to target cRNA (also known as

anti-sense RNA)

General Process Overview

1. Using PCR or other techniques, synthesize probes specific to sequence(s) of interest and attach them to array; another option is to purchase premade arrays specific to gene(s) of interest

2. Isolate mRNA from cells of interest3. Transform this mRNA into cDNA by reverse

transcriptase using fluorescently labeled nucleotides in order to create labeled cDNA

• for RNA microarrays reverse transcriptase is omitted and the mRNA is labeled

4. The labeled cDNA is washed over the microarray and will bind to any matching probes

General Process Overview

5. Excess cDNA is washed away, leaving only hybridized sequences.

6. Microarray is excited with lasero The labeled,

hybridized cDNA will fluoresce at different intensities

7. Microarray is scanned and quantifiedo The brighter/ more

concentrated the color, the more DNA is present

General Process Overview

Attaching the Probe to the Array

• DNA fragments are chemically tethered to glass, plastic, nylon, or silicon biochips (also known as DNA chips)

• This is done in different ways according to which type of microarray is being used. The two common types are:o Spotted arrayso In situ synthesized arrays

Spotted Arrays• The nucleotide sequences of interest are

generated by PCR and “spotted” onto the array surface by a robot.

• Sequences are ~1 kb in length• Relatively low costs and easy to synthesize

for smaller gene sets

http://www.youtube.com/watch?v=3ZXq_aDfSB8

In situ Synthesized Arrays

• Also known as photolithographic arrays• Sequences of interest are synthesized directly on

the array surface

• Sequences are ~30-70 bp long more specific binding and more probes per slide

• Much more expensive than spottedo The individual masks must be designed and manufactured.

This is the most tedious step, and is what makes the process pricey.

o To combat this cost, Digital Light Processing (DLP) has been developed; this process uses a set of movable micromirrors to apply light to certain areas on the array. This computerized process bypasses the need for the masks.

http://www.youtube.com/watch?v=MuN54ecfHPw(view up to 30 second mark)

In situ Synthesized Arrays

Protecting groups

Nucleotides

Types of Signal Detection

One color• Each sample labeled with same

fluorophore• Expression is compared between multiple

arrays• Usually used with in situ; also requires

twice as many arrays $$• Less prone to error• Simpler design

Two color• Control and experimental samples labeled

with two different fluorophores• Hybridized on same array

• Allows for direct comparisons• Cheaper (also commonly used with

spotted)• Can compare ratio of two genes to

determine if their expression levels are related

• Sensitive to error, more complex design

Applications

• Gene discovery• Drug discovery

o Individualized treatments

• Toxicological research

• Gene expression studies• Disease diagnosis• Pathogen analysis

o Rapid genotyping

Microarray Pros and Cons

Pros• Many pre-made probes

available in market• Prior knowledge to gene

sequence is not required

• Large cDNA works well with hybridization

• Spotting technology is readily available

• Process is fast and flexible

• Can be used for all organisms

• Study multiple genes at once

Cons• Contamination potential

extremely high• Cross-hybridization with

similar, repetitive gene families can create false positives

• Not extremely quantitative

• In situ can become very expensive

• Many possible errors during scanning due to background noise

In Summary:Complete process overview:

http://www.youtube.com/watch?v=3jX_08zdYCE

Microarray technology is a very powerful tool used for many applications and if often paired with PCR. This field is continuing to grow, leading to cheaper prices as well as increasingly specialized

applications.