Dna microarray, Role of nucleic acid .

Role of nucleac acid microarray

1. Role OF NUCLEIC ACID MICROARRAY Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY PRESENTED BY Kushal R 2 nd Sem M Pharm SUBJECT INCHARGE Dr Veeresh P Veerapur M Pharm., Ph D Professor Dept Of Pharmaceutical Quality Assurance

2. CONTENTS Introduction Principle of Microarray Nucleic A cid Microarray Principle of Nucleic Acid Microarray cDNA based microarrays Applications of DNA Microarray In Drug Discovery Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

3. INTRODUCTION Microarray technology is a general laboratory approach that involves binding an array of thousands to millions of known nucleic acid fragments to a solid surface, referred to as a C hip . Microarray technology can be used for a variety of purposes in research and clinical studies, such as measuring gene expression and detecting specific DNA sequences (e.g., single-nucleotide polymorphisms). A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

4. Principle of Microarray The principle behind microarray is that complementary sequences will bind to each other. The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent markers are attached to these fragments. These are then allowed to react with probes of the DNA chip. Then the target DNA fragments along with complementary sequence bind to DNA probes. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

5. The remaining DNA fragments are washed away. The target DNA pieces can be identified by their Fluorescence emission by passing a laser beam. A computer is used to record the pattern of fluorescence emission and DNA identification. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

6. NUCLEIC ACID MICROARRAY Nucleic Acid Microarray also called as DNA microarray. DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene, these slides are referred to as Gene chips or DNA chips . The DNA molecules attached to each slide act as probes to detect gene expression, which is also known as the Transcriptome or The set of messenger RNA (mRNA) transcripts expressed by a group of genes. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

7. Principle of Nucleic Acid Microarray The basic principle behind the DNA microarray is Nucleic acid hybridization . In this process, two complementary strands of a DNA are joined together by hydrogen bonds to form a double-stranded molecule. This helps researchers to compare and analyze the DNA or RNA molecules of identical sequences. Types of DNA Microarray cDNA based microarrays Oligonucleotide based microarrays Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

8. Requirements of DNA microarray: DNA chip Fluorescent dyes Ex – Cyanine 3[Cy3] & Cyanine 5[Cy5] Fluorescent labelled target/sample Probes – Ex - Oligodb, Oligopicker, Sarani & Probewiz Scanner Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

9. cDNA based microarrays: cDNA is used for the preparation of chips. It is a high throughput technique. It is highly parallel RNA expression assay technique that allows quantitative analysis of RNAs transcribed from both known and unknown genes. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

10. Steps involved in cDNA based microarray: Sample collection Isolation of mRNA Creation of labeled cDNA Hybridization Collection and analysis Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

11. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

12. Sample collection:  A sample can be any cell/tissue that we desire to conduct our study on. Generally, 2 types of samples are collected, i.e. healthy and infected cells, for comparing and obtaining the results. Isolation of mRNA: The extraction of RNA from a sample is performed by using a column or solvent like phenol-chloroform. mRNA is isolated from the extracted RNA leaving behind rRNA and tRNA. As mRNA has a poly-A tail, column beads with poly-T tails are employed to bind mRNA. Following the extraction, buffer is used to rinse the column in order to isolate mRNA from the beads. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

13. Creation of labeled cDNA: Reverse transcription of mRNA yields cDNA. Both the samples are then integrated with different fluorescent dyes for the production of fluorescent cDNA strands which allows to differentiate the sample category of the cDNAs. Hybridization: The labeled cDNAs from both the samples are placed on the DNA microarray which permits the hybridization of each cDNA to its complementary strand. Then they are thoroughly washed to remove unpaired sequences. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

14. Collection and analysis: Microarray scanner is used to collect the data. The scanner contains a laser, a computer and a camera. The laser is responsible for exciting the fluorescence of the cDNA, generating signals. The camera records the images produced at the time laser scans the array. Then computer stores the data and yields results instantly. The data are now analyzed. The distinct intensity of the colors for each spot determines the character of the gene in that particular spot. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

15. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

16. Applications of DNA Microarray In Drug Discovery Gene expression and profiling : It is employed in detection of the presence and absence of specific genes, compare genes from different sources and to know how the external genes affect the external stimuli. It is used to detect the gene expression pattern of different diseases such as cancer. It is also helpful in studying the effect of certain treatments, diseases and developmental stage on gene expression. Disease Diagnosis:  It helps the researchers to investigate about different diseases such as heart diseases, infectious diseases, mental illness and cancer. Due to the advancement in microarray technology, it makes very easy to know the types of cancer on the basis of gene acting in tumor cells. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

17. Gene Discovery:  This technology is used in identification of new genes and their functions as well as their expression level in various conditions. Drug Discovery:  It is extensively applied in Pharmacogenomics. The comparative analysis of genes help to identify specific proteins which is produced by diseased cell resulting to synthesize drug that combat with these proteins and help to reduce their effects.  DNA microarrays can be utilized to identify compounds that target certain genes or pathways and to evaluate possible therapeutic candidates for their influence on gene expression. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

18. Research:   It is beneficial in conducting different researches related to toxicogenomic studies and nutrigenomic studies. It is also used to reveal the presence of antibiotic resistance genes and to study microbial pathogenicity. LIMITATIONS of DNA Microarray The initial high cost for the synthesis of gene specific primers, fluorescence used in labeling is the major limitation. The shelf life of the chips -are not very long. The analysis of results become complex as too many results at a time. Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

19. REFERENCES https://www.slideshare.net/SanjaySinhmar/dna-microarray-31210546 https://www.slideshare.net/Haddies/dna-microarray-dna-chips https://www.genome.gov/about-genomics/fact-sheets/DNA-Microarray-Technology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467903/ https://www.ncbi.nlm.nih.gov/probe/docs/techmicroarray/ Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY

20. Thank you Department of Pharmacology SREE SIDDAGANGA COLLEGE OF PHARMACY