Nucleic Acids & Transcription Quiz

Nucleic Acids & Transcription

• Tutorials now follow the same schedule as lectures
• SciLearn answers are not posted online; discuss with a TA during Q&A sessions
• Instructor office hours: Send questions by Wednesday, 3 pm
• Midterm results (tentative) will be released next week
• Questions/concerns should be directed to the assessment email, not the general TA email

Nucleic Acid Detection

• Nucleic Acid detection methods include Southern, Northern, and Western blots
• Southern blot: DNA detection; uses restriction enzymes for fragment separation
• Northern blot: RNA detection
• Western blot: Protein detection; uses acrylamide gel, denatures polypeptides
• Probes (single-stranded DNA or RNA) hybridize with the target, allowing for detection in blotting techniques. Probes are labelled to amplify signals.
• Nitrocellulose or Nylon is used as a solid phase support to transfer the separated nucleic acids from the gel for easier handling and better access to probes/antibodies

Detecting Nucleic Acids

• Complex mixtures of macromolecules are separated based on size using agarose gel electrophoresis
• Probes specific to the target nucleic acid are used
• Hybridization with a specific probe identifies nucleic acids of interest
• Nitrocellulose or nylon membranes can bind to the nucleic acid to aid in easy handling and access for probes or antibodies
• Target Detection on membrane is visualized through autoradiography

Probe Generation

• Oligonucleotides are short, single-stranded or double-stranded DNA/RNA molecules
• Oligonucleotides can be labelled for detection
• Polynucleotide Kinase (PNK) can be used to label oligonucleotides
• Polymerase Chain Reaction (PCR) can be used to produce radio- or fluorescence-labeled DNA probes

Transfer of Nucleic Acids to Solid Support

• Nucleic acids are separated according to size on agarose gels
• Separated molecules are transferred to a solid support (e.g., nitrocellulose) for handling and access to probes/antibodies
• DNA is fragmented to easier handling

Southern Blot: Restriction Fragment Length Polymorphisms (RFLPs)

• Differences in DNA sequences can be detected by the presence of fragments of different lengths
• these differing fragment lengths arise from DNA digestion with specific restriction enzymes
• RFLPs can be used for diagnostic or relatedness purposes

Northern Blot: Differential gene expression

• RNA is heated to eliminate secondary structure before electrophoresis
• RNA samples are transferred to a solid support (e.g., nitrocellulose)
• Visualized by detecting hybridization of probes to specific RNA molecules
• This method is used to study spatiotemporal information about gene expression patterns. Differences in RNA expression levels under different conditions (e.g, different tissues, development stages) are revealed

qRT-PCR: Another way to determine mRNA expression

• mRNA is converted to cDNA using reverse transcription
• Fluorescent dyes are incorporated into the cDNA signal, which is measured as a function of amplification time
• The faster the reaction, the more cDNA, and the more mRNA is present.

RNA-Seq: Determining mRNA expression

• RNA-Seq is a technique for determining global gene expression in a sample
• Complete transcriptome, the set of all RNA transcripts, is determined for an individual or population of cells
• RNA is converted to cDNA and amplified
• Using next-generation sequencing (NGS), specific sequences on the cDNA are identified
• Provides a complete picture of global mRNA expression profiles

Eukaryotic Transcription (RNA Pol II)

• Eukaryotic cells have 3 types of RNA Polymerases (I, II, III)
• RNA polymerase II transcribes a range of genes to synthesize mRNA, snRNAs and other small RNAs.

Transcription (DNA to RNA)

• Initiation: RNA polymerase binds to promoter; DNA is locally denatured; first phosphodiester bond is formed.
• Elongation: RNA polymerase moves away from the start site; DNA template is copied
• Termination: RNA polymerase encounters a transcription stop site, releasing the completed RNA strand and dissociating from the DNA

Transcription Regulation

• Promoters are regions of DNA where proteins bind to start transcription
• Enhancers are DNA elements that affect transcriptional ability of RNA Polymerase
• DNA loops allow enhancers to affect transcription regions far from them. Up/Downstream refers to the direction from the transcription start site

Transcription

• Eukaryotes have 3 well-conserved, multimeric RNA polymerases
• RNA Pol I is responsible for almost all rRNA of the cell
• RNA Pol II synthesizes mRNA and certain types of small noncoding RNAs
• RNA Pol III synthesizes tRNAs and certain ribosomal components

Promoter Start Sites

• The TATA box is located upstream (10-35 bp) of the transcription start site (TSS)
• TBp (TATA-binding protein) binds to the minor groove of DNA and disrupts double helix structure, aiding Pol III transcription

Linker Scanning Mutagenesis

• Used to determine promoter elements - introduce mutations in reporter constructs
• Compare expression of reporter mRNA from each mutant to identify required subregions of the promoter region for activation of transcription