BIO1334 L5 ELE PCR Cloning Engineering PDF

Genetic tools: The polymerase chain reaction and Sanger sequencing Manipulating DNA and gene cloning Dr Yusra Siddiqui [email protected]  Intended learning outcomes After the lecture (and lecture consolidation) you should: Under...

Genetic tools: The polymerase chain reaction and Sanger sequencing Manipulating DNA and gene cloning Dr Yusra Siddiqui [email protected]  Intended learning outcomes After the lecture (and lecture consolidation) you should: Understand the principles of PCR and the steps and components involved Know how to visualise PCR products Understand the principle of Sanger sequencing Understand how these lab techniques relate to our knowledge of DNA synthesis Understand the key tools used in recombinant DNA technology (i.e. chopping up bits of DNA and putting them together in new combinations) Give an example of the use of genetic engineering  DNA replication – DNA synthesis RECAP – LAST LECTURE DNA polymerase dNTPs Template DNA Primer - DNA synthesis 5’ to 3’ direction 5′ 3′-OH 3′ 3′ 5′  DNA synthesis in vitro Can these components be brought together in the lab, in a tube to DNA synthesis in vitro? Is it possible to perform multiple rounds of a polymerase-catalysed DNA synthesis? If so, could DNA be amplified in vitro?  Polymerase chain reaction (PCR) Amplification of DNA Ingredients for PCR: Template DNA Primers dNTPs Buffer (Mg2+) Taq polymerase* *DNA strands are separated by heat Hot spring, Yellowstone National Park, Wyoming http://bioinfo.bact.wisc.edu/themicrobialworld/LAHT/b27.html  Polymerase chain reaction (PCR) 94o C ~72oC ~50- 65oC 1. Denaturation 2. Annealing 3. Extension double DNA primers bind to at 72oC: strand melts DNA and optimum open polymerase temperature Essential Cell Biology, Fifth Edition attaches and for polymerase Copyright © 2019 W. W. Norton & Company starts copying and extension Polymerase chain reaction (PCR)  Polymerase chain reaction (PCR) (21) (22) (23) Amplification is exponential After 30 cycles = 230 copies = 1 073 741 824 copies  Polymerase chain reaction (PCR) Only DNA between primers is amplified Cycle 1 Specific sequences can be amplified from a complex mixture of DNA. The ends of the amplified fragment are Cycle 2 defined by the 2 primers. The primers are ~20 bp single single stranded DNA (oligonucleotides) They are chemically synthesised (~15p/base) Cycle 3  Analysis of DNA – agarose gel electrophoresis 1 2 3 4 Stain DNA e e e e pl pl pl pl m m m m with Sa Sa Sa Sa fluorescent dye for detection by UV exposure e.g. Separation of DNA by agarose gel electrophoresis : ethidium Electric current is applied to the gel bromide DNA moves to + electrode because it is negatively charged through gel depending on: Conformation (shape) – linear/circular/supercoiled Size – smaller fragments move through the gel faster than large ones  Analysis of DNA – agarose gel electrophoresis Stain DNA with fluorescent dye for detection by UV exposure e.g. ethidium bromide Standards of known sizes “ladder” Sample 1 1 2 Sa le 3 4 ~550 bp e e e pl pl pl 3000 bp p m m m m 1500 bp Sa Sa Sa Sample 2, 3, 4 500 bp ~750 bp Analysis of DNA – agarose gel electrophoresis This is the sort of thing we will be doing in the practi  Uses of PCR Any research uses in which a specific piece of DNA needs to be amplified For example: - Detection of pathogens in water, clinical samples et c. - DNA sequencing - Diagnosis of genetic disorders - Prenatal diagnosis - Analysis of ancient DNA - Genetic fingerprinting - Forensic analysis  Limitations of PCR! 1. Sequence information is required to design 2 primers 2. Limit on length of amplified fragment 3. Potentially high error rate eg. Taq polymerase = ~10-4 4. Very sensitive to exact reaction conditions, so not easily quantified 5. Tiny amounts of contaminating DNA will also be amplified  PCR products can be sequenced Eg. Sanger sequencing  The principle of Sanger sequencing DNA polymerase dNTPs +ddATP, ddTTP, ddGTP, ddCTP Template DNA Primer DNA synthesis 5’ to 3’ direction 5′ 3′-OH 3′ 3′ 5′  The principle of Sanger sequencing DNA polymerase dNTPs +ddATP, ddTTP, ddGTP, ddCTP Template DNA Primer DNA synthesis 5’ to 3’ direction 5′ 3′-OH 3′ 3′ 5′ The principle of Sanger sequencing Manipulating DNA and gene cloning  Plasmids as tools Bacterial genomes are typically a Bacteria often circular chromosome also harbour plasmids which are small extrachromosoma l circles of DNA  Cutting DNA- Restriction enzymes DNA can be cut by restriction endonucleases – enzymes extracted from bacteria Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  Cutting DNA How often might we expect this enzyme, HindIII, to cut the E. coli genome? 5’ - - AAGCTT - - 3’  Cutting DNA 5’ - - AAGCTT - - 3’ This sequence will occur every ¼*¼*¼*¼*¼*¼* or 1 in 46 bp = 1/4096 bp The Escherichia coli genome is ~4.6 Mb long or 4.6 x 106 bp Therefore the E. coli genome will have approx. 4.6 x 106 4096  1123 sites  Joining DNA DNA ligase - Allows two DNA molecules to - Forms phosphodiester bonds be joined – Recombinant DNA - Requires ATP Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company  Insertion of a DNA fragment into a bacterial plasmid Essential Cell Biology, Fifth Edition Molecular Biology of the Cell (© Garland Science ) Copyright © 2019 W. W. Norton & Company  Gene cloning Introduce recombinant plasmid into bacterial cell Will be replicated Cell will divide Clone of cells Recover DNA for analysis Gene cloning (Can also clone genes by insertion into bacteriophage DNA vectors) Figure 8-39 Molecular Biology of the Cell (© Garland Science)  Transgenics Genes between species Genetic code is “universal”* It is possible to take a gene from one organism and express it in another  Example: production of insulin Govender, T., Naicker, T. Insulin as a catalyst to recombinant DNA technology. Nat Catal6, 454–455 (2023). https://doi.org/10.1038/s41929-023-00976-7  Key takeaway points PCR can be used to amplify specific areas of DNA Sanger sequencing can be used to sequence the nucleotides of amplified DNA Restriction endonucleases cut DNA at specific (restricted) sequences. Ligases join two pieces of DNA. DNA can be inserted into plasmids in the lab for amplification or to make a library. Using genetic engineering we can answer biological questions and  Recommended reading material: Relevant areas of one of the core text books or other general genetics text book – for example… Essential cell biology Chapter 10 – Analysing the structure and function of genes iGenetics Chapter 10 – Recombinant DNA technology (relevant sections)