Approaches to Addressing Biological Questions at the Molecular Level Genetic Approach Studying/Altering genes to learn about function of gene products in vivo Forward Genetics: Find mutant and identify what causes phenotype Reverse Genetics: Find gene of interest and create mutant (using introduction of additional sequences: transgenes) and observe phenotype Transgenes: Genetic material transferred to an organism which may alter phenotype Silence Genes: miRNA and siRNA complementary to target mR

Cloning: asexual reproduction of genetically identical organisms (generation of organisms contains specific DNA) The Process of Cloning Restriction Enzyme Digest to cut vector (plasmid BAC YAC) and fragment, ligation (T4 DNA ligase) to form recombinant molecule, ligation products introduced into host cells via transformation Isolation of Genomic Fragments by Library Screening Gene Libraries give a means to isolate specific cloned fragments, library is created by ligating each of the restriction

Vector nucleic acid molecule used to carry and replicate sequences of interest in a host organism Replicate Independently of chromosomal DNA: Allow high copy # inside cell and ability to replicate Unique cloning sites: Short segment of DNA with RE sites, allows researcher to ligate DNA specifically Selectable markers: To detect the transformation Easily recovered from host cell: Allows isolation of recombinant from host cell for reuse or remove Plasmid Vectors Circular, supercoiled DNA molecules

Extraction and Purification of Nucleic Acids Collect tissue containing nucleic acid of interest Use fresh material, or frozen quickly to maintain quality and limit degradation by nucleases which release during homogenization Homogenize tissue and lyse cells EDTA: Chelates Mg and inactivates nuclease activity since Mg is cofactor for nucleases SDS: Solubilizes membrane and lyse cells Bacterial: Sonification, freeze thaw cycles, enzymes, high pressure Animal/Plant: Homogenizers, Yeast bead breaker

Genomic Library Total gDNA from a single organism, stored in a population of identical vectors Isolate pieces of genomic DNA (gene of interest) Isolate fragments adjacent to a specific fragment Gene mapping and genome sequencing Promotor, introns, intergenic region, non-expressed genes, DNA → DNA cDNA Collection of cloned cDNA fragments inserted into host cells, contain only mRNA sequence Estimate transcript abundance Identify novel genes, splice variants, amino acid sequence Isolate coding regi

PCR allows for isolation of nucleic acids and analysis (can study rare genes by amplifying small amounts of DNA and generate specific fragments) also useful for cloning and RE site can be added to ends via primers Requires: Template, Forward & Reverse Primers, dNTPs, DNA ligase, DNA polymerase, Buffer (Mg) Initial denature: 2m, 95 (ensure ss), Denaturation: 30s, 95, Anneal: 30s, Tm - 5 (Tm=2*(AT)+4*(GC) Extend: 1m per 1000 bp, 72, Elongate: 5m, 72 Leave at 4oC Annealing Conditions: Optimized for

Probes can be designed to bind specific or group of sequences with a region in common, method to label probe depends; type of template available, whether strand specific probe is needed, whether very sensitive probe is needed. Screening Libraries with Gene Probes: Hybridization Libraries must be screened to find cone carrying fragment of interest, use a modified DNA/RNA fragment or an antibody, strategy depends on what you know about target sequence Bound probe must be detectable & quantifiable

Two early techniques for DNA sequencing Maxam Gilbert (now only for footprint experiments) Sanger (common) Sanger Uses 2’ 3’ ddNTP, single stranded DNA is the template for reaction that is terminated by ddNTP, invitro Can sequence the insert of plasmid without purifying from the plasmid Add primer complementary to sequence before the insert, long enough to be specific to target Carry out invitro DNA synthesis with ddNTP and labelled nucleotide In each tube carry out reaction starting with same t

Most sequencing methods rely on shotgun sequencing: do a partial digest of fragment, get reads and using computational methods overlap the reads and get the entire sequence Clone by Clone Approach Using genetic mapping techniques, sequences large genome by ordering sub clones Many copies of the genome are fragmented in partial digest and the large fragments are cloned into BACs and amplified, clones are purified for further analysis and completely digested, chosen to produce a patter of small f

Targeted Approach Transcript is known, and we want to know abundance/localization Northern Blot In-situ Hybridization Semi-quantitative PCR Untargeted Approach looking for differences amongst transcripts profiling/monitoring EST Abundance Transcriptome Sequencing Microarrays Subtractive Hybridization Genes respond to more than 1 factor and can be expressed constitutively, spatially, temporally Constitutive: expressed at all times in every cell but may change level of expression (actin) Tempora

Analysis or protein abundance: biochemical approach → protein purification by chromatography → obtain amino acid sequence and develop DNA probes to screen cDNA for a clone Proteome: Protein complement of a cell Protein Purification Must purify protein of interest from a mixture, commonly by column chromatography based on size (gel filtration), charge (ion exchange), antibody affinity etc. SDS Page Separation of denatured proteins based on size, useful to evaluate purte/composition of protein mix

When studying protein of unknown function, common approach is to do bioinformatics, and then consider how strong is the interaction, does it require other components, specific conditions determine subcellular localization and search for interactors, Pull Down Assay using Epitope Tagged Proteins Affinity column for epitope tag on a protein or an extract invitro Tag added to protein to tether or link the protein to column, protein extracts prepared from cells that express the linked protein normal

This analysis is done after cis elements of promoter have been suggested but still don’t know what they are, few fragments with potential cis elements but consider the notion that many transcriptional factors bind as dimers Electrophoretic Mobility Shift Assay Label DNA fragment with potential cis element labelled radioactively because ethidium bromide will not detect Mix with protein with potential trans factor Electrophorese the fragment alone, product of DNA + protein, and DNA + protein + ant