Biotech Techniques PDF

Basic Techniques in Biotechnology  PCR principles PCR is an in vitro technique that takes a specific sequence of DNA of small amount and amplifies it to be used for further testing P Polymerase DNA polymerase is used in the process C Chain...

Basic Techniques in Biotechnology  PCR principles PCR is an in vitro technique that takes a specific sequence of DNA of small amount and amplifies it to be used for further testing P Polymerase DNA polymerase is used in the process C Chain One reaction’s products are next one’s substrate R Reaction Transformation of a set of substances Chemistry Nobel Prize Kary Mullys - 1993 PCR Procedure  PCR essentials DNA Sample  PCR procedure Temperature (ºC) 1- Denaturation Time (min)  PCR procedure Temperature (ºC) 2-Primer Annealing Time (min) *+, 𝑇𝑚 𝑝𝑟𝑖𝑚𝑒𝑟𝑠 = 81,5 + 0,41 (%GC)− − % 𝐦ismatch 𝐍  PCR procedure Temperature (ºC) 3-Elongation Time (min) This cycle (denaturation-annealing-elongation) is repeated ≈30 times, doubling the amount of target DNA each time. 9 𝐷𝑁𝐴 𝑐𝑜𝑝𝑖𝑒𝑠 = 𝐷𝑁𝐴. x 2/º 123456 30 Cycles à x10  Primer design Ø Efficiency and specificity of a PCR rely on a good primer design Rules: Ø Length: 15-30 nucleotides. Ø 40-60% of C+G content. C or G must be on 3’ Ø Tm of the pair (Forward and Reverse) should not differ a lot Ø Repetitive or complementary sequences must be avoided  Primer design Repetitive sequences lead to primer dimers or unproductive PCR 5’-NNNNNNNNNTATA-3’ 5’-NNNNNNNNNTATA-3’ 3’-ATATNNNNNNNNN-5’ 5’-NNNNNNNNNTATA-3’ PRIMER DIMER 3’-NNNNNNNNNTATA-5’ 5’-TATANNNNNNNNNTATA-3’ 3’-NNNNNNNNNTATA-5’ 3’-NNNNNNNNNATAT-5’ NO EXTENSION  Primer design Repetitive sequences lead to primer dimers or unproductive PCR 5’-NNNNNNNNNGCA 5’-NNNNNNNNNGCATGC-3’ 3’-CGT 5’-NNNNNNNNNNNNNN-3’ HAIRPIN UNWANTED PRODUCTS We will do a practical exercise later…. Basic Techniques in Biotechnology  Gene cloning is the process in which a gene of interest is located and copied (cloned) out of all the DNA extracted from an organism GENETIC ENGINEERING: Isolation Modification Introduction  Genetic Tools: Key Tools Enzymes ACTION Enzyme MODIFICATE Methylases CUT Endonucleases PASTE Ligases FOSFATE ADITION Quinases FOSFATE ELIMINATION Phosfatases SYNTHESIS Polymerases DEGRADATE Exonucleases First protocol General protocol Cloning Vector Elements Cloning Vector Elements A vector must have the necessary nucleic acid sequences to ensure that it can be replicated within the host cell Ori is the sequence on the vector where its replication is initiated using the host cell’s replication machinery (DNA polymerase and other enzymes) Generally, ori sequences are rich in A and T, and low in G and C nucleotides. A-T pairs have two hydrogen bonds while G-C pairs have three. Thus, AT rich sequences at the ori region separate easily, making the DNA single-stranded, which is important for the DNA replication to occur smoothly A vector replicon comprises the ori and all other sequence elements that regulate its replication Cloning Vector Elements Cloning Vector Elements Cloning Vector Elements We will do a practical exercise later…. Basic Techniques in Biotechnology Bioproduction of a recombinant protein in certain amount for different purposes (research, biomedical, pharmaceutical nutritional,… ) maintaining the same properties as the native protein  How? "In vitro”: transcription and translation of “In vivo”: It involves the complete the gene encoding the protein using expression of a DNA coding sequence that enzyme systems and/or crude cell extracts. is introduced into a growing cell lineage. AIM: Obtaining milligrams of the recombinant protein  Sequence elements for Expression Vector expression of cloned DNA fragment Elements Expression Vector Type of organism Vector Characteristics Modifications Modifications E.g. proteins in pharma Bacteria Plasmids (