BioM1 Recombinant DNA-based molecular techniques PDF 2020-2021

Recombinant DNA-based molecular techniques (part I) DNA cloning Prof. Mamoun Ahram What is DNA cloning? DNA cloning is a technique that allows for: amplifying a DNA segment into many, many copies in a biological system. expressing a gene inside a biological system such as bacteria, human cells grown...

Recombinant DNA-based molecular techniques (part I) DNA cloning Prof. Mamoun Ahram What is DNA cloning? DNA cloning is a technique that allows for: amplifying a DNA segment into many, many copies in a biological system. expressing a gene inside a biological system such as bacteria, human cells grown in labs, animals, or even the human body as a whole. It usually involves: The formation of a recombinant DNA composed of a vector (a carrier of the gene or the DNA segment of interest; usually a bacterial plasmid) and a gene that encodes a protein or a non-coding RNA using restriction endonucleases. Insertion into the cell(s). Restriction endonucleases Endonucleass are ezymes that degrade DNA within the molecule. Restriction endonucleases: Bacterial enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments. Palindromic sequences The sequences recognized by restriction endonucleases—their sites of action—read the same from left to right as they do from right to left (on the complementary strand). Types of cuts by restriction endonucleases Restriction enzymes cut DNA in two different ways: Blunt: enzymes cut at the same position on both strands giving blunt-ended fragments. Staggered (off-center): enzymes cut the two DNA strands at different positions generating sticky or cohesive ends. The DNA restriction fragments would have short single-stranded overhangs at each end. Zoom into the sticky ends DNA ligase It covalently joins DNA ends (example, restriction fragments) by catalyzing the ATP-dependent formation of phosphodiester bonds between the 3’-hydroxyl group of one strand and the 5’-phosphate end of another strand. Cloning Cloning means that you make several copies of one thing. A clone is a genetically identical population, whether of organisms, cells, viruses, or DNA molecules. Every member of the population is derived from a single cell, virus, or DNA molecule. How do we clone a DNA molecule? a DNA fragment of interest is inserted into a DNA carrier (called a vector) that can be replicated. The resulting DNA molecule is what is known as a recombinant DNA molecule. The procedure is known as recombinant DNA technology, which is part of genetic engineering. Using plasmids as vectors Bacterial plasmids are considered excellent vectors that are used for cloning (cloning vectors) or expression (expression vectors). These are natural bacterial circular DNA that is not part of the main circular DNA chromosome of the bacterium. A plasmid exists as a closed circle and replicates independently of the main bacterial genome. Features of plasmid cloning vectors Plasmid cloning vectors must have the following three components: Their own origin of replication (OriC) that allows them to replicate independently of the bacterial chromosome. Aselectable gene such as an antibiotic resistance gene that allows for selecting for/against the cells that have them. A restriction site that allows for insertion of the DNA segment of interest into the plasmid. The making of a recombinant DNA Both DNA fragments (the DNA to be cloned and a vector) are cut by the same restriction endonuclease that makes DNA fragments with same sticky-ends hybridize (anneal) to each other, when mixed. A DNA ligase is added to “close” the plasmid. 12 Cloned DNA Cell clones 13 Overview of gene expression Expression vectors Expression vectors contain additional sequences: Promoter sequences upstream of gene to be inserted, Ribosomal binding sequences (ShineDalgarno [SD] sequences), A transcription termination sequence. The protein is expressed and purified. Examples: insulin, growth hormone, plasminogen activator, erythropoietin A gene How do we select for human mRNA? The power of reverse transcriptase (part 1) The “many types of RNA” challenge The “poly-T primer” solution A poly-T primer How do we deselect introns? The power of reverse transcriptase (part 2) The “intronic” challenge The “reverse” solution Challenges of protein expression in bacteria No internal disulfide bonds No post-translational modification (example: glycosylation) Protein misfolding Protein degradation Solution: use a eukaryotic system such as yeast Protein tagging and creation of protein hybrids Proteins can be "tagged" A protein-encoding gene is cloned in a special vector containing a tag gene producing a protein with an extra sequence of amino acids called tags. These tags allow easy protein purification and detection. Direction of transcription Post-protein tagging…1) Affinity chromatography https://www.youtube.com/watch?v=8_7cdfNO7OY Post-protein tagging…2) Immunoprecipitation https://www.youtube.com/watch?v=41T1Az_EsrE Post-protein tagging…3) Gel electrophoresis (SDS-PAGE) into the wells of a gel to separate proteins according to size https://www.youtube.com/watch?v=MILiO1XnuqQ by staining proteins that look like “bands” Post-protein tagging…4) Immunoblotting https://www.youtube.com/watch?v=EAKSr4Eclyw Major protein tags Name Amino acids Detection Purification FLAG DYKDDDDK antibody FLAG peptide Green fluorescent proteins (GFP) ~220 aa protein antibody or fluorescence None Glutathione S transferase (GST) 218 aa protein antibody glutathione HA YPYDVPDYA antibody HA peptide Poly-His HHHHHH antibody nickel, imidazole Myc EQKLISEED antibody Myc peptide V5 GKPIPNPLLGLDST antibody V5 peptide His tag The addition of six histidines to a protein would allow for purification using breads with bound nickel ions. Clone Express Purify Analyze Purification of GST-tagged proteins Glutathione S transferase (GST) Production of a recombinant protein Gene A Gene B Protein A Protein B Recombinant gene Recombinant protein Production of a recombinant protein…The power of domains Gene A Domain Gene B Domain Protein A Domain Domain Protein B Recombinant gene Recombinant protein A protein domain is a compact region (or part) of the protein's polypeptide chain that: 1) has a defined three-dimensional structure, 2) is self-stabilizing, 3) folds independently from the rest. 4) can be disconnected from the protein and, yet, maintains its structure and function. Proteins can have several domains. GFP-tagged proteins Green Fluorescent Protein (GFP) allows for protein detection rather than for purification purposes. A world of possibilities