PRESENTATIONON

DNA CHIPS

CONTENTS What is DNA chip? Principle uses of chips Major technologies Background of DNA DNA nomenclature DNA chip design Applying DNA principle to chips Why we need to know about DNA What you need to know about DNA Micro arrays DNA and researchers History Application Principle Micro arrays and bio-informatics Uses and types Alternative technologies

WHAT IS A DNA CHIP?

A DNA microarray (also commonly known as DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

Principle uses of chips

Genome-scale gene expression analysis Differentiation Responses to environmental factors Disease processes Effects of drugs

Detection of sequence variation Genetic typing Detection of somatic mutations (e.g. in

oncogenes) Direct sequencing

Major technologies cDNA probes (> 200 nt), usually produced by

PCR, attached to either nylon or glass supports

Oligonucleotides (25-80 nt) attached to glass support

Oligonucleotides (25-30 nt) synthesized in situ on silica wafers (Affymetrix)

Probes attached to tagged beads

BACKGROUND OF DNA Genetics started in 1866 . Genes were linked to DNA. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes.

These probes can be a short section of a gene or other DNA element.

Probes are used to hybridize a cDNA or cRNA sample.

Dna nomenclature

• Double Helix• Four Bases

Dna chip design

Probe selection Non-redundant set of probes Includes genes of interest to project Corresponds to physically available clones

Chip layout Grouping of probes by function Correspondance between wells in microtitre

plates and spots on the chip

Applying DNA principle to chips

• Chips are designed to either “sequence” or decode genetic strands, or to find genetic matches.

• HYBRIDIZATION• The array provides a medium for matching known and unknown

DNA samples based on base-pairing (hybridization) rules. The two strands basically combine automatically if correct matching has occurred.

Why you need to know about DNA Chips•They are astonishingly powerful.• They allow scientists to quickly and inexpensively do experiments they could only dream about just a few years ago--like tracking how the expression of every single gene in the human genome changes in response to an experimental variable. •They are cheap and fast.•Chips make it possible to find out not just the average human genetic sequence, but about your personal genetic sequence.•They have consumer applications•They are becoming big business.• Analysts estimate that the annual sales of DNA chips will exceed 1 billion dollars within just a few years.

What you need to know about DNA Chips

•“DNA chip” is slang for DNA microarray. • DNA chips are a revolutionary technology. They speed up research, helping scientists understand the primary sequence of the human genome.• They will also allow doctors to get important genetic information from individual patients and thus choose the best treatments.

•Microarrays can have tens of thousands of spots. This means they can look for tens of thousands of DNA sequences all at once. •A sequencing array is made of many different short DNA sequences. •Researchers use these to find the sequence of an unknown bit of DNA. •A researcher chops the unknown sequence into short bits, sees where the bits bind on the array, deduces the sequences of all the short unknown bits, then reassembles the overlapping sequences into one long sequence.

Researchers love DNA chips because they give a huge amount of information, fast, at low cost. Doctors will soon learn to love them because there are many times when a doctor would like to know something about a patient's genes (such as whether the patient is likely to respond well to a certain drug). When the price comes down enough, microarrays will likely become routine tools in the doctor's office.

DNA AND RESEARCHERS

WORKING

• Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known DNA sequence.

• The first reported use of this approach was the analysis of 378 arrayed lysed bacterial colonies each harboring a different sequence which were assayed in multiple replicas for expression of the genes in multiple normal and tumor tissue.

• This was expanded to an analysis of more than 4000 human sequences with computer driven scanning and image processing for quantitative analysis of the sequences in human colonic tumors and normal tissue and then to comparison of colonic tissues at different genetic risk.

History

CONT…..

These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays for gene expression profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997

The use of a collection of distinct DNAs in arrays for expression profiling was also described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon.

DNA microarrays have enabled biology researchers to conduct large-scale quantitative experiments. This capacity has produced qualitative changes in the breadth of hypotheses that can be explored.

It has become the dominant mode of use, changes in the transcription rate of nearly all the genes in a genome, taking place in a particular tissue or cell type, can be measured in disease states, during development, and in response to intentional experimental perturbations, such as gene disruptions and drug treatments.

The response patterns have helped illuminate mechanisms of disease and identify disease subphenotypes, predict disease progression, assign function to previously unannotated genes, group genes into functional pathways, and predict activities of new compounds.

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Applications

CONT..

Directed at the genome sequence itself, microarrays have been used to identify novel genes, binding sites of transcription factors, changes in DNA copy number, and variations from a baseline sequence, such as in emerging strains of pathogens or complex mutations in disease-causing human genes.

They also serve as a general demultiplexing tool to sort spatially the sequence-tagged products of highly parallel reactions performed in solution

PRINCIPLE

The core principle behind microarrays is hybridization between two DNA strands.

The property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.

A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands.

After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized.

Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization.

Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot.

Microarrays use relative quantization in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position

PRINCIPLE

Microarrays and bioinformatics

The multiple levels of replication in experimental design (Experimental design)the number of platforms and independent groups and data format(Standardization)the treatment of the data (Statistical analysis)accuracy and precision (Relation between probe and gene)the sheer volume of data and the ability to share it (Data warehousing)

The advent of inexpensive microarray experiments created several specific bioinformatics challenges

Many types of arrays exist and the broadest distinction is whether they are spatially arranged on a surface or on coded beads:The traditional solid-phase array is a collection of orderly microscopic "spots", called features, each with thousands of identical and specific probes attached to a solid surface, such as glass, plastic or silicon biochip (commonly known as a genome chip, DNA chip or gene array). Thousands of these features can be placed in known locations on a single DNA microarray.The alternative bead array is a collection of microscopic polystyrene beads, each with a specific probe and a ratio of two or more dyes, which do not interfere with the fluorescent dyes used on the target sequence

Uses and types

Alternative technologies

Advances in massively parallel sequencing has led to the development of RNA-Seq technology, that

enables a whole transcriptome shotgun approach to characterize and quantify gene expression. Unlike microarrays, which need a reference genome and

transcriptome to be available before the microarray itself can be designed, RNA-Seq can be also be

used for new model organisms whose genome has not been sequenced yet

REFERENCES Statwww.epfl.ch/davision/…week03.ppt Clifton.mech.northwestern.edu www.seminarsonly.com www.Dnawikipedia.com

PRESENTATIONMADE BY

Punit Jainpunitjain5885.blogspot.com