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DNA Microarray Technology

DNA Microarray Technology. Nucleic acid hybridization Construction of (human) genomic and cDNA libraries Polymerase chain reaction (PCR) Expression microarrays Work-flow of microarray experimentation. Nucleic Acid Hybridization.

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DNA Microarray Technology

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  1. DNA Microarray Technology • Nucleic acid hybridization • Construction of (human) genomic and cDNA libraries • Polymerase chain reaction (PCR) • Expression microarrays • Work-flow of microarray experimentation

  2. Nucleic Acid Hybridization • DNA denaturation can be achieved by high temperatures or high pH. By slowly cooling or lowering the pH DNA can renature • The nucleic acid hybridization techniques make use of the pairing properties of (A, T), (A, U) and (G, C) • DNA probe := oligonucleotide or a short (10-1000 nucleotides) single-stranded DNA • Cloning (two possible meanings in biology) • The act of making many identical copies of DNA molecule • The isolation of a particular stretch of DNA from the rest of the cell’s DNA.

  3. Genomic and cDNA libraries • Genes can be isolated by cloning but dealing with 3 billion (human) nucleotides is not an easy task. This can be avoided by breaking up the total genomic DNA into smaller, more manageable pieces. • The process of creating genomic libraries: • Total DNA is extracted from a tissue sample or a culture of (human) cells • DNA is cut up into fragments by restriction nuclease treatment • Each fragment is cloned using bacterial plasmids and utilizing DNA ligase • The collection of cloned DNA fragments is known as a DNA library

  4. Genomic and cDNA libraries • ?: how one can find a particular gene in this huge genomic library • If the sequence of complimentary DNA is known, one could make a probe and use it to identify that particular gene by exploiting the properties of hybridization • If, on the other hand , the sequence of the gene is not known, one can use protein sequencing to identify a few of the amino acids coded by that gene. Then, by reversing the genetic code one can synthesize a suitable DNA probe and use it to identify the clone(s) • It is possible though, that for a given gene several clones my be identified, as no single clone might contain the entire gene.

  5. Genomic and cDNA libraries • For many applications, it is advantageous to obtain a clone that contains only the coding sequence of a gene, i.e. no intron DNA. This process is known as the creation of a cDNA library • Steps in creating a cDNA library • Starting from the mRNA that is expressed in a particular tissue or cell culture, construct the complimentary DNA using reverse transcriptase • Degrade the mRNA copy and use the single-stranded cDNA to produce a double-stranded cDNA copy of the original mRNA • Clone the so obtained cDNA molecules

  6. Polymerase Chain Reaction (PCR) • A synthetic procedure that can be used to selectively replicate a given nucleotide sequence quite rapidly and in large amounts from any DNA that contains it. • Steps in performing PCR • Heat up the double-stranded DNA to denaturate it • Primers are hybridized on the two strands to mark the beginning of the regions of DNA to be amplified • DNA polymerase and deoxyribonucleosidetriphosphates are added so that complementary DNA originating from the primers can be synthesized. Thus the original DNA is amplified by a factor of 2

  7. Expression Microarrays • All three levels in the central dogma – DNA, RNA, and protein – interact. Thus, it is not possible to fully separate them, and information from all the levels must be combined to fully understand the cellular control • Expression microarrays := grids of thousands of different single-stranded DNA molecules or oligonucleotides attached to a surface to serve as probes. • cDNA microarrays • Synthetic oligo microarrays

  8. Expression Microarrays • Basic procedure involving expression microarrays • Extract RNA from cells • Convert RNA into single-stranded cDNA • Attach fluorescent labels to the different cDNAs • Allow the single-stranded cDNAs to hybridise to their complementary probes on the microarray • Detect the resulting fluor-tagged hybrids via excitation of the attached fluors and image formation using a scanning confocal microscope • Relative RNA abundance is measured via measurement of signal intensity which is obtained by image processing and statistical analysis

  9. cDNA microarray

  10. Synthetic Oligonucleotide Arrays • Affimetrix chip • Short (25 mers) sequences of nucleotides obtained by solid-state DNA synthesis are printed using photolithography on the slide • 25-mers can peir with many mRNAs. Thus, for every mRNA of interest, there is a set of non-overlapping probes to provide a sequence-specific detection • The measurement for a specific mRNA is obtained via averaging across the set of probes • Two sets of probes are used: perfect match (PM) and mismatch (MM) where only one nucleotide does not match the target sequence

  11. Hardiman’s paper – important points Read the entire paper and get a general idea what the paper is about. It is important to know the 3 different types of microarrays (cDNA, short and long oligos). There are two important parts of Table 1 labeled as "Advantages" and "Disadvantages" respectively. The sections "Accuracy of array experiments", "Which platform is best?", "Comparison of microarray expression platforms",  and "Outlook and conclusions"  should be read carefully and you should know the key points the author makes in those sections. However, I will not expect from the students to know the details from those sections. The "Highlights" box on pg. 500 could serve as a guide of what is important.

  12. Work-flow of Microarray Experimentation • Hybridize array. • Collect results. • Normalize. • Analyze.

  13. Data Normalization • Correct for systematic bias in data. • First step in comparing data across the microarrays from an experiment. • Approaches: - Housekeeping genes - Spiked-in controls - Global median - Total intensity - LOWESS correction

  14. Variation in data • Wanted : Across experimental conditions. • Unwanted : Chip, slide, hybridization, imaging. • Multiple hypotheses testing. • ANOVA. • Classification. • Clustering. • Network modeling.

  15. Experimental design • Array design • Samples • Hybridizations • Measurements • Normalization

  16. Course Project

  17. Formulate the question Organizing and cleaning data Interpretation of results Normalize data Analyze data

  18. The question • Experimental design: 3x2x2 factorial design • How many animals? • How many arrays? • Replicates?

  19. Organize, Clean, and Normalize Data • Label data • Remove labels • Quality of data: missing, low or good • Normalization: choice of normalization makes a difference

  20. Analyze Data • Differentially expressed genes. • Classification. • Clustering. • Feature selection.

  21. S(ingle)N(ucleotide)P(olymorphism) Arrays

  22. SNP array: a type of DNA microarray which is used to detect polymorphisms within a population. • Single nucleotide polymorphismn (SNP): a variation at a single site in DNA, the most frequent type of variation in the genome. There are about 5-10 million SNPs in the human genome. SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research.

  23. Basic Principles The convergence of DNA hybridization. Fluorescence microscopy. Solid surface DNA capture.

  24. Components The array that contains immobilized nucleic acid sequences or target. Labeled probes. A detection system that records and interprets the hybridization signal.

  25. Applications Markers for Mendelian diseases with complex traits efficiently. Personalized drug development. Studying the Loss of heterozygosity (LOH). LOH is a form of allelic imbalance. Has the potential in cancer diagnostics as LOH is a prominent characteristic of most human cancers.

  26. High-throughput assays technologies DNA • Polymorphism • SNP arrays • Mutation • CGH arrays • Loss of Heterozigosity RNA • Expression levels • Microarrays • SAGE Protein • Relative abundance • Mass Spectrometry • Modification • ChIP2chip • Activity

  27. SNP • A single nucleotide (A,T,C, G) DNA sequence alteration … ACGGCTAA … … ATGGCTAA … • It must occur in at least 1% of the population. • SNPs make up ~90% of all human genetic variation. • Occur every 100 to 300 bases along the 3-billion-base human genome. • 2/3 of SNPs are replacement of C with T. • Evolutionary stable.

  28. C(opy)N(umber) and LOH • Genomic instability defining feature of cancer cells (mutations, heterogeneity, …). • DNA copy number changes is one of the hallmarks of the genetic instability common to most human cancers • Loss of Heterozigosity of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. • “two-hit hypothesis”: mutation + LOH = loss of function

  29. Measuring genetic variation • Affymetrix SNP arrays. • Represent gene w/ set of 10 probe pairs (per allele): • Each probe (oligonucleotide) is a 25-long sequence of bases characteristic of one gene.

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