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2012.03.14 - Bio 1202 Lecture Review Notes.docx

3 Pages

Course Code
Biology 1202B
Gardiner/ Murphy

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Biology Lecture Review Notes Lecture 8 Application of DNA Technologies  The basis of many of these methods are denaturing the DNA, then trying to find a complementary sequence  Total RNA extracted from a cell o 80% rRNA o 10-20% tRNA o 1-5% mRNA  mRNA is unstable to work with - RNases are everywhere & degrades RNA quickly o RNases – enzymes that cut RNA o mRNA (no introns – only exons) is smaller than gDNA so a PCR fragment can contain potentially more important information than a piece of gDNA (some exon but lots of intron sequence) o Sometimes it is advantageous to work with a copy of the mRNA, but in a stable DNA form (i.e. cDNA)  cDNA Synthesis makes a copy of the mRNA o Oligo DT = sequence of 23 T’s o Most mRNA has a poly A tail (so we are using the complementary T’s to bind to the poly A tail and synthesis will occur from the poly A tail) o Or could use a gene-specific primer to do 5’ to 3’ synthesis o Or could use a random primer – they can bind anywhere, along any transcript, generating cDNA o When making cDNA synthesis, we need an enzyme (reverse transcriptase) o The fragments made from the reverse transcription are an RNA/cDNA hybrid (top is RNA, bottom is cDNA) o RNase enzyme removes the unstable RNA to generate a single stranded cDNA o This cDNA can now go through all the techniques we can do, and it is stable  Microarray - allows us to examine a tissue and look at all the transcripts at one time o Rather than looking for the presence of one specific transcript, you can look for the presence of 60 thousand transcripts at once o Use 2 different samples (one control group and an experimental group) o Extract single stranded RNA and convert it into cDNA o Use dye to differentiate between the two samples  Cy3 = green dye (control group)  Cy5 = red dye (experimental group) o Mix the two single stranded together and allow them to try and find complementary sequences on the membrane (microarray)  There is competition between the red and green cDNA o The cDNA in each circle on the array is different (blades of grass) o Computer is used to analyze the samples o Green = Cy3 only (gene expressed in normal tissue) o Red = Cy5 only (gene expressed in experimental tissue) o Yellow when Cy3=Cy5 (gene expression equal in normal & experimental tissue) o When there are black spots – there is nothing bin
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