Class Notes (839,081)
Canada (511,183)
Biology (6,826)
Lecture

2012.03.14 - Bio 1202 Lecture Review Notes.docx

3 Pages
110 Views

Department
Biology
Course Code
Biology 1202B
Professor
Gardiner/ Murphy

This preview shows page 1. Sign up to view the full 3 pages of the document.
Description
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
More Less
Unlock Document

Only page 1 are available for preview. Some parts have been intentionally blurred.

Unlock Document
You're Reading a Preview

Unlock to view full version

Unlock Document

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit