BioMG 3320 1st Edition Lecture 5Outline of Past LectureI. Comparing Genomes II. History of Genome SequencingIII. Gel Electrophoresis of Nucleic AcidsIV. DNA Sequencing: Gilbert MethodV. DNA Sequencing: Sanger MethodVI. Illumina SequencingOutline of Current LectureI. Recombinant DNAII. Cloning VectorsIII. PCRIV. DNA CloningV. Building a Physical Map of the GenomeCurrent LectureI. Recombinant DNA- Recombinant DNA is a new DNA sequence that is created by combining existing sequencesThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Restriction Endonucleases (type II) are enzymes that recognize SPECIFIC sequences in DNA and cleave the DNA within the recognition sequence, allowing recombinant DNA to form-Creates a molecule with single-stranded end that can be combined with another molecule (also known as a “sticky” end because the two strands will stick together- Biology of restriction endonucleases:-Bacteria possess many pathways to transfer DNA between cell species-Restriction endonucleases perform a protective role by digesting foreign DNA-the foreign DNA is restricted-Host DNA is protected from cleavage by site-specific DNA methylases; methylation of the recognition sequence interest with function of the restriction endonucleases- the host DNA is modified-Pair of enzymes: a methylase and a restriction endonuclease which have overlapping recognition sequences-Methylation of DNA is a recurring theme across biology- There is a large variety of restriction enzymes that form different types of ends on the fragments-EcoRI is common and forms staggered endsII. Cloning Vectors- Cloning vectors contain DNA of interest; allows for replication and purification of E.coli- Need plasmid (relatively small circular double-stranded DNA molecules that are competent to replicate which are found endogenously in bacteria)- To clone vectors:-Take cleaved DNA-Insert cleaved DNA into vector (plasmid) using DNA ligase for propagation -Introduce recombinant DNA into host cell by transformation-Cells containing the plasmid can be selected by growth on the agar plates that contain growth media; they will be the only cells to survive, producing a resistant colonyIII. PCR- PCR is a method to amplify a specific DNA sequence - It consists of: -Denaturing the DNA strand-Anneal: basepair between template and primer-Extension: Allow polymerization- PCR utility is greatly enhanced by:-Chemically synthesized DNA oligonucleotides-PCR machines-Variety of thermostable DNA polymeraseIV. DNA Cloning- DNA cloning refers to propagating identical copies of a segment of DNA through recombinant DNA technology- Process:-Open vector with restriction enzymes; prepare insert with restriction enzymes-Ligate insert into vector-Introduce DNA into bacteria: transformation-Grow bacteria selecting for cells that receive vectorV. Isolation of Protein of Interest- This is a way to get large quantities of one protein- There is a strong non-covalent interaction between GST (a protein) and glutathione (small molecule) ligand- Process:-Take gene for target protein-Take gene for GST and fuse together-Place into E.coli-Prepare cell extract containing fusion protein as part of the cell protein mixture-Add protein mixture to column. Glutathione anchored to medium binds GST tag and other proteins flow through column, so that the protein of interest is isolatedVI. Building a Physical Map of the Genome- Fragment genomic DNA into very large pieces and clone Bacterial Artificial Chromosomes (BAC). Then, purify each BAC-clone, digest with restriction enzymes and run on gel. Finally, determine the order of the BAC-clones across entire genome; identifya set of BACS that overlaps and covers the genome.-This is a library clonic vector that covers the entire genome-Resulted in a near-complete genome: some chromosomes had gaps. Final closing of gaps took years- The other method is known as shotgun sequencing:-Take genome and break it up-Tell computer to put the pieces together-Assembles by placing overlapping DNA sequences