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Lectures 18 and 19 .docx

4 Pages

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Biology 1202B
Brenda Murphy

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1 Application of DNA Technologies: cDNA Synthesis, Microarrays, Genetic Variation Basics of DNA Technology  Denature dsDNA to to ssDNA) & Complementary strands will re-anneal (hydrogen bonding to form double helix again)  Double-stranded DNA  Denature it with high temperature or increased salt concentrations  becomes a single-stranded DNA  Single-stranded DNA  Renaturation  Renatured DNA (will be double-stranded again) o Renatured single strands of DNA can renature to give the duplex form Recall RNA  Total RNA extracted from a cell o 80% rRNA o 10-15% tRNA o 1-5% mRNA  Interested in mRNA, and since it is very small you have to be very careful  mRNA is unstable (due to RNases) to work with o Ribonuclease everywhere and degrades RNA quickly  mRNA is smaller than gDNA so 1kb PCR fragment (using cDNA template) can contain potentially more important information (just exons) than a piece of gDNA (some exon but lots of intron space)  Sometimes its advantageous to work with a copy of the mRNA (cDNA) but in a stable DNA form o DNA more stable nucleic acid than RNA cDNA Synthesis (In Vitro) Makes a Copy of the mRNA - When we expose to chemicals - The T‘s will bind to the A‘s and you will get synthesis - Any gene that has been transcribed and given in an mRNA has the potential to be picked up (as long as there is a poly-A tail) 2 Expression Microarray Analysis of Gene Expression Levels  Purpose – Can be used in various experiments including comparing the levels of gene expression in two different tissues. The power of the technique is that the entire set of genes in a genome can be analyzed simultaneously  A DNA microarray can be used to compare gene expression in normal cells and cancer cells in humans o mRNAs are isolated from each cell type, and cDNAs are made from them, incorporating different fluorescent labels: green for one cDNA, red for the other  The two cDNAs are mixed and added to the DNA chip, where they hybridize with any complementary probes  A laser locates and quantifies the green and red fluorescence—can see which genes are expressed in the cells, and for those that are expressed, you can quantify differences in gene expression between the two cell types  Preparation of the microarray o Each area contains unique single stranded cDNA sequence from a specific transcript  Transcripts are tissue specific (ie. Blood will not be the same as brain)  They are also time specific (ie. Birth, puberty etc.)  Preparation of the Specimen Sample o Obtain normal (reference) and cancer cells (experimental) o Isolate total RNA o Convert to single-stranded cDNA and level it with dye  Normal = green = Cy3  Cancer = red = Cy5 o mRNA labeled with Cy3 and Cy5 competing for complementary sequence on microarray
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