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PTT 104 Introduction to Biotechnology Lecture 3 Techniques in Biotechnology Recombinant DNA & PCR Madam Noorulnajwa Diyana Yaacob Course Outcome CO 2: Ability to demonstrate important recent advances in methods and applications of biotechnology with regards to microorganisms and plants. Biotechnology today • Focuses on DNA Deoxyribonucleic Acida double-stranded helical molecule that stores genetic information for the production of all the organism’s proteins What Is the Structure of DNA? • DNA Is Composed of Four Nucleotides • DNA Is a Double Helix of Two Nucleotide Strands • Many people contributed to the discovery, but Francis Crick and James Watson (and Maurice Wilkins) got the Noble prizes. Hydrogen Bonds • Hydrogen bonds hold certain nitrogenous base pairs together – A bonds with T, G bonds with C – Bonding bases called complementary base pairs • Ladder-like structure of the two DNA strands are twisted into a double helix What Is the Structure of DNA? • Hydrogen Bonds Between Complementary Bases Hold the Two DNA Strands Together • The Order of Nucleotides in DNA Can Encode Vast Amounts of Information 5 end 3 end 3 end Figure E9-6 Biology: Life on Earth 8/e ©2008 Pearson Prentice Hall, Inc. 5 end Recombinant DNA • DNA produced by joining segments of DNA from different sources • eg. To produce human insulin, scientists have combined bacterial plasmid DNA + human DNA Tools for Producing Recombinant DNA Restriction enzymes: enzymes that cleave the DNA double helix at specific nucleotide sequences Use of the Restriction Enzyme Bam H1 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ Results in sticky end 5’— G 3’— C C T A G sticky end G A T C C — 3’ G — 5’ Tools for Producing Recombinant DNA Vector: carrier of DNA; can be virus or plasmid Plasmid: extrachromosomal, independently replicating, small circular DNA molecule Producing Recombinant DNA Mix together Add DNA Ligase restriction enzyme Treat source DNA with restriction enzyme Treat plasmid DNA with same enzyme Many recombinant DNA molecules are produced, each with a different piece of source DNA restriction enzyme Transform bacterial cells Each bacterial cell carries a different recombinant plasmid Tools for Producing Recombinant DNA Probe: sequence of DNA that is complementary to the gene of interest; Used to locate a copy of the gene by hybridization AGCTTAGCGAT TCGAATCGCTA Denature DNA by heating Add Probe Probe Binds to gene AGCTTAGCGAT AATCGC TCGAATCGCTA Building a DNA Library Applying Your Knowledge 1. 2. 3. 4. Probe Clone Plasmid Restriction Enzyme A. An enzyme that cleaves DNA at specific sequences is a __________ . B. A sequence of DNA that is complementary to the gene of interest is a _________. C. A small, independently replicating DNA molecule is a ___________. Polymerase Chain Reaction • PCR is a technique that is used to amplify a sample of DNA from miniscule amount of DNA (ex., DNA from a crime scene, archaeological samples, organisms that can’t be cultured). Who developed PCR? • PCR was developed by Kary Mullis. • Kary Mullis is a scientist and surfer from Newport Beach, California. • He won a Nobel Prize in Chemistry in 1993 for the development of PCR. • He was working for Cetus Corporation in the 70’s and received $10,000 bonus for the idea. How is PCR used? • Medical Diagnosis: To detect and identify the causes of infectious diseases from bacteria and viruses. • Genetic testing: To determine whether a genetic mutation has been passed on (ex. cystic fibrosis). • Evolutionary study: To gather archaeological samples and analyzed for similarities/differences. • DNA fingerprinting: To profile DNA from blood, hair, and skin cells for criminal identification and forensics Stages of PCR PCR is divided into 3 stages: 1. Denaturation 2. Anneal 3. Extension What is a primer? • A primer is a short oligonucleotide which is the reverse complement of a region of a DNA template. • It would anneal to a DNA strand to facilitate the amplification of the targeted DNA sequence. oligonucleotide Primer Selection variables • • • • • • Primer length Melting Temperature GC content Hair-pin loop Self-dimerization Cross-dimerization Primer Length • Should be between 18 – 25 bases. • The longer the primer, the more inefficient the annealing. • If primers are too short, they will cause non-specific annealing and end up amplifying nonspecific sequences. Melting Temperature • Formula (18-25 bp range): – Tm = 2(A+T) + 4(G+C) • The forward and reverse primers should be having similar Tm, or else amplification will be less efficient. • Melting Temperature should be between 55ºC and 65ºC. GC Content • GC% = (G + C) / length of seq * 100% • The base composition should be in the range of 45% to 55%. • Poly G’s or C’s can result in non-specific annealing. Hairpin Loop • Primers with hairpin loop may interfere with annealing to the template by forming partially double-stranded structure. Self-dimerization • Primers may form inter-primer homology with its own copies. Cross Dimerization • Forward and Reverse primers may hybridize to form primer-dimer. Algorithm for primer design Input DNA sequence Input the start and end of central region Input the length of primers N 55-65oC Y GC content 45-55% Y Hairpin and selfdimerization List of acceptable primers N Y N N Cross Dimerization Y Excluded primers Tm: Polymerase Chain Reaction (PCR) Amplifies a specific region in the DNA Used for identification, especially if the amount of DNA is small Uses repeated cycles of heating to denature DNA and cooling to synthesize new DNA Involves the use of ---Taq polymerase (withstands heat) ---primers to begin synthesis Polymerase Chain Reaction: One PCR Cycle 90 °C 50 °C Taq Polymerase Original Doublehelix DNA Separate DNA Strands 72 °C Primer Primers & Taq polymerase bind DNA synthesized