Deoxyribonucleic Acid DNA

Transcription

1 Introduction to BioMEMS & Medical Microdevices DNA Microarrays and Lab-on-a-Chip Methods Companion lecture to the textbook: Fundamentals of BioMEMS and Medical Microdevices, by Prof., Deoxyribonucleic Acid DNA Groton Public Schools Space-filling model of the DNA double helix. James D. Watson and Francis Crick, who discovered the structure of DNA in Chromosomes & Genes Chromosome 1 Access the National Health Museum (left) National Center for Biotechnology Information (right) 1

2 Eukaryotic Gene Regulation Evolution-textbook.org; Modified from Seet, BT et al. Nat. Rev. Mol. Cell. Biol. 7: , 2006 Overview of Compounds Studied Casado-Vela, J. Screening of protein-protein and protein-dna interactions using microarrays: applications in biomedicine. Advances in Protein Chemistry and Structural Biology, Volume 95, 2014 DNA Microarrays 2

3 Array Structure Capability Measurement of tens of thousands of genes at a time. It is possible to obtain near-comprehensive expression data for individual, tissues, or organs in various states. Comparisons are possible for transcriptional activity across different tissues in the same organism, across neighboring cells of different types in the same tissue, across groups of patients with and without a particular disease or with two different diseases. Direct measurement of the activity of the genes involved in a particular mechanism or system. Formats Microarray analysis allows simultaneous study of genes and gene products, including DNA, mrna and proteins. There are basically two formats: cdna microarrays (clone-based): A cdna microarray is an orderly arrangement of spots of cdna clones (from a library) printed onto a solid matrix such as glass, nylon or silicon. Oligonucleotide-based microarrays : Photolithographic in situ synthesis. Used for gene expression, genotyping (single nucleotide polymorphisms or SNPs), and resequencing. 3

4 Resequencing The Resequencing Array is used for identifying DNA in a sample. Resequencing means that when the DNA is sequenced, it can be compared with an already established sequence database for the DNA of interest. The probes that are used are made of segments of DNA 25 bases long, but the probes are used to basically sequence the DNA in the sample rather than identify a specific RNA or SNP. Gene Expression Gene expression microarrays are tools that tell how much RNA (if any) a gene is making. They use the natural chemical attraction, or hybridization, between DNA on the array and RNA target molecules from the sample based on complimentary base pairs. Only RNA target molecules that have exact complementary base pairs will bind to the probes. Microarrays can measure the expression of every known human gene. Single Nucleotide Polymorphisms Small differences in a DNA sequence can have major impact on health. Using microarrays, it is possible to scan the whole genome and look for genetic similarities among a group of people who share the same disease. A genotyping microarray may look for up to 100,000 SNPs or more. DNA samples from the patient may be obtained from any biological sample, since DNA is the same in all cells of the body (unlike the RNA sampling for gene expression). 4

5 Oligonucleotide Microarray Methodology RNA is extracted from a sample using PCR, allowing it to be more easily detected on the array. When the RNA is copied, biotin caps are attached to each strand that will later act to bind fluorescent molecules that are washed over the array. Note that different cells in the body have different amounts of RNA. Hybridization The sample is washed over the array for hours to allow hybridization to occur. This process allows the chemical bonding of the DNA probes with the matching RNA fragment. Fluorescence Measurement The amount of fluorescence in a given feature correlates with the amount of RNA that was present in the original sample. If there is no fluorescence over a feature, then there is no matching RNA present. Lighter areas represent increased expression. Image courtesy of Affymetrix 5

6 Output The Affymetrix Scanner work station and GeneChip Array output is data from an experiment showing the expression of thousands of genes on a single GeneChip: Fabrication of Microarrays Silanation of Silicon Surface Image courtesy of Affymetrix 6

7 Probe Synthesis Inspection, Dicing and Packaging Polymerase Chain Reaction An in vitro method of replicating small DNA sequences into millions of copies over a short period of time. Nanomolar quantities of DNA can be replicated within a few hours. PCR may be used for genetic testing in disease diagnosis, monitoring response to treatment, and tissue typing. A typical PCR requires: Two oligonucleotide primers; A thermally stable DNA polymerase; Supply of free nucleotides; and A small amount of DNA sample that contains the sequence of interest. The DNA fragment of interest must be known, so that short DNA primer fragments can be synthesized in advance, and are complimentary to the 3 end of each sample stand. 7

8 Denaturing and Annealing Steps A. Desired sequence or target region to amplify. B. Denaturing by heating 95 C C. Annealing by cooling to 60 C, and binding of primers. Carey, FA, Advanced Organic Chemistry 5 th Ed., 2002 Synthesis and Denaturing D. End of Cycle 1 The primer is extended in its 3 direction as it adds nucleotides that are complementary to the original DNA strand. E. Denature again and prime. Carey, FA, Advanced Organic Chemistry 5 th Ed., 2002 Elongation and Sequence Amplification F. Elongation of the primed polynucleotide fragments completes the second cycle and gives 4 DNAs. End of Cycle 2 G. Among the eight DNAs formed in the third cycle are two having the structure shown. This structure increases disproportionately in the succeeding cycles. Carey, FA, Advanced Organic Chemistry 5 th Ed.,

9 DNA Amplification Results Cycle Number Total Number of DNAs Number of DNAs Containing Only the Target Region ,024 1, ,048,566 1,048, ,073,741,824 1,073,741,746 Carey, FA, Advanced Organic Chemistry 5 th Ed., 2002 A CMOS IC qpcr LOC for POC Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, Utilizes a Single-Photon Avalanche Diode The cross-section of a SPAD CMOS sensor Albert H. Titus, et al. CMOS Photodetectors in Photodiodes - World Activities in Ed. by Park, JW, pub. by InTech,

10 Heaters and Sensors Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, Fully Encapsulated PCR Chip Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, qpcr Thermal Cycling Profile Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14,

11 Characterization of the SPAD Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, Real-Time Quantitative PCR Data Norian, H. et al., An integrated CMOS qualitative-polymerasechain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, A PCR + Microarray Device Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013)

12 Lab-on-a-Chip Cross-Section Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013) Process Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013) Summary (1) Chromosomes, genes and gene regulation. (2) DNA Microarrays (3) Methodology (4) Fabrication (5) Polymerase Chain Reaction (6) A CMOS IC qpcr LOC for POC (7) A PCR + Microarray Device 12