Studying Genes

• Studying genes: organism’s genome is one complete copy of information required to specify an organism (in humans 23 chromosomes, about 3.2 billion nucleotides)
• Less than 1.5% of human DNA is protein coding (~21,000 genes); if you include introns which are very large, 30% of genome contains genes
• In molecular biology we want to study the function of one or more genes
• Genes are isolated for study by cloning them into vectors that permit their selection and amplification.
• A gene or genomic segment is cut out of a chromosome with a restriction enzyme (or PCR amplified) and ligated into a vector. The recombinant vector is transferred into a host cell and is amplified in this transformed cell.
• Gene cloning relies on an arsenal of enzymes made available by advances in molecular biology, including restriction endonucleases, DNA ligase, DNA polymerase, and reverse transcriptase.
• Transformation is a natural process that we use in the lab to  introduce recombinant DNA into bacteria that act as factories (you need heat shock/CaCl or electroporation)
• Important cloning vectors include plasmids, bacterial artificial chromosomes, and yeast artificial chromosomes. BACs and YACs allow the cloning of very long DNA segments.
• We have engineered plasmids to contain polylinkers: multiple recognition sequences for restriction endonucleases
• Plasmids are naturally found in nature, small, extrachromosomal DNA segments; replicated independently from bacterial chromosomes; often have natural antbiotic resistance
• When we work with plasmids in the lab we have to be careful and always cut them first if we want to truly size plasmids (plasmids can exist in many forms and supercoiled forms travel faster through gels)
• FEATURES OF A LAB PLASMID: ori; antibiotic resistance; polylinker with unique sites; transcription regulation elements if you need to express it
• Antibiotics commonly used as selective agents: most antibiotics affect translation with the exception of ampicillin which inhibits cell wall formation
• Genes or other DNA segments can be amplified by the polymerase chain reaction.
• PCR uses: thermostable DNA polymerase; primers, building blocks and heating and cooling cycles
• We can use PCR to amplify DNA  for cloning or to detect the presence of viruses or identify genetic diseases or look at the expression levels of different genes
• qPCR can be used to quantitate copy number of genes or specific sequences and especially for virus detection
• Two methods of qPCR: either sybr green and forward and reverse primer or qPCR uses fluorescent probe and fluorescence quencher
• How to read a qPCR result (Ct is the threshold cycle number, the earlier the Ct, the more abundant our starting material)
• RNA needs to be revere transcribed before PCR can take place and because many viruses are RNA based, we need to do RT-qPCR to detect them
• Cloned genes can be altered. A gene sequence can be changed, sequences deleted, or sequences added. All changes affect the protein or RNA product of the gene. This can be accomplished with site directed mutagenesis at the level of PCR
• Sanger method for sequencing, know how it works: requires sequencing primer and DNA polymerase, dNTPs and ddNTPs
• Cloned genes can be expressed to amplify protein production (cloning vector also has regulatory sequences of transcription)
• ANY organism can serve as a host to express proteins