Nucleic Acids & Transcription RR6-8 - BIOL200 PDF
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Uploaded by CongenialCarnelian9331
McGill University
Holly J
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Summary
This document is a set of lecture notes on nucleic acids and transcription. It covers various topics such as detecting nucleic acids, methodologies like Southern Blot, Northern Blot, and Western Blot, probe generation using PCR, and transfer of nucleic acids. The document also discusses methodologies like linker scanning mutagenesis and eukaryotic transcription, including initiation, elongation, and termination.
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Nucleic Acids & Transcription RR6-8 BIOL200 | Holly J Tutorials now follow the same schedule as the lectures! Housekeeping SciLearn: Answers to problem sets will NOT be posted on MyCourses. If the Tuesday/Friday sessions are full, you can dis...
Nucleic Acids & Transcription RR6-8 BIOL200 | Holly J Tutorials now follow the same schedule as the lectures! Housekeeping SciLearn: Answers to problem sets will NOT be posted on MyCourses. If the Tuesday/Friday sessions are full, you can discuss the answers with a TA during the post-tutorial Q&A session Instructor Office Hours: Send in questions by 3pm the Wednesday before Midterm Results: (Tentatively) Released next week Any questions/concerns should be directed to [email protected] NOT the general TA email! Nucleic Acid Detection Detecting Nucleic Acids Southern Blot: Detecting DNA Northern Blot: Detecting RNA Western Blot: Detecting Proteins Detecting Nucleic Acids Probe Generation How can we detect nucleic acids? 1. Complementary 2. PCR to make oligonucleotide labelled with radio/fluorescence labelled Polynucleotide Kinase (PNK) DNA probes Oligonucleotide : Short single/double stranded DNA/RNA molecules Transfer of Nucleic Acids to Solid Support How can we detect nucleic acids? 1. Nucleic acid is separated DNA is very long, according to size on agarose so it needs to be fragmented gel 1 2. Molecules are transferred to a solid state support 2 (nitrocellulose) which allows for easier handling of separated molecules and easier accessibility to probes/antibodies Transfer of Nucleic Acids to Solid Support How can we detect nucleic acids? 3. Bound, separated molecules 4 can be hybridized to 3 probes/antibodies 4. Only bands corresponding to probe/antibody will be visible after washing the support and imaging with autoradiograph Southern Blot: Restriction Fragment Length Polymorphisms Differences in DNA sequences that can be detected by the presence of fragments of different lengths after digestion of DNA with specific restriction enzymes Can be used for diagnostic purposes, or to confirm relatedness Northern Blot: Differential gene expression Can be used to determine spatiotemporal information about a gene’s expression qRT-PCR: Another way to determine mRNA expression 1. Convert mRNA to cDNA using reverse transcription - Dye in the solution incorporates itself into the cDNA so that the more cDNA is made, the stronger the signal 2. Fluorescence signal is measured, based on how long it takes each sample to get to its PCR plateau - The more cDNA (and therefore the more mRNA) there is, the less time it will take (lower Ct value) Specific mRNA expression RNA-Seq: Another way to determine mRNA expression Global gene expression in a sample -> Looking at the transcriptome (set of all RNA transcripts in an individual/a population of cells) Global mRNA expression Eukaryotic Transcription (RNA Pol II) Transcription DNA to RNA INITIATION Polymerase binds to the promoter sequence, locally denatures the DNA and catalyzes the first phosphodiester linkage (Clamp domain is open) Transcription DNA to RNA ELONGATION Polymerase moves away from transcription start site and flips into a processive configuration (Clamp domain of RNA Pol II shuts) Transcription DNA to RNA TERMINATION Polymerase recognizes the stop site and releases the RNA, then dissociated from DNA Transcription DNA to RNA + 1 DNA looping allows for enhancers to act on regions linearly far away from them Transcription DNA to RNA Clamp Domain: Needed for stability and processivity – rotates on a hinge when DNA enters RNA Pol II, and shuts during elongation to anchor DNA Carboxyl Terminal Domain (CTD): Is phosphorylated during transcription, promoting interactions of transcription related protein Transcription Regulation Promoter Start Sites Linker Scanning Mutagenesis Determining promoter elements Introduce small mutations in the control region Compare expression of the reporter mRNA from each Each mutation will be cloned mutant into a plasmid that has a By “lining up” mutations, you can determine which regions reporter mRNA of the control region are necessary for transcription
