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Lecture 3

BIOLOGY 1A03 Lecture 3: THEME 4

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Rosa Da Silva

Module 3 - Applications of DNA replication UNIT I: REPLICATION IN A TUBE KEY COMPONENTS IN IN VITRO REPLICATION (1985) • Karly Mullis developed the ability to amplify DNA in tube via the Polymerase Chain Reaction (PCR) technique • Allowed copy/amplification of millions of copies of DNA from small starting samples • Shed light on diagnosis of genetic defects, detection of viral DNA, production of large amounts of DNA from fossils containing trace amounts, and linking individuals to DNA during forensic investigations SETTING UP PCR • Sample of DNA is placed into a tube containing a buffered solution with essential ions/salts, and a single-stranded DNA primer (15-30 nucleotides) • Primers have complementary binding to specific regions of the template DNA and serve as starting points • Since this is a cell-free system, deoxyribonucleotides (dNTPs) are also added and utilized during replication • Instead of using enzymes to unwind/stabilize DNA, tubes are placed in thermocycler machines which heat/cool to facilitate DNA replication over numerous cycles • TAQ POLYMERASE: enzyme which is tolerant to high temperatures that is added to catalyze polymerization of each daughter strand per cycle ONE ROUND OF REPLICATION • Each cycle includes three key stages: denaturation, annealing, and extension • This cycle brings about a chain reaction that produces an exponentially growing population of identical DNA depending on type of primers • Primers are designed that bind/anneal to their complementary sequence on either side of the DNA of interest on the template strands DENATURATION: double helix is unwound by the high temperature of the reaction mixture ANNEALING: thermocycler will cool the solution to allow annealing of two primers (on opposite strands at each end of the target sequence) to their complementary sequences on the DNA template on either side of the DNA sequence of interest EXTENSION: Taq polymerase extends/polymerizes the daughter strands using four dNTPs starting from the primers and moving in a 5'-3' direction Each complete cycle results in two helices containing the desired target sequence portion of the template DNA REPEATED PCR CYCLES AMPLIFY DNA • With each cycle, the number of replicated molecules with the same sequence as the parent template doubles (2 , n= number of cycles) • After the first cycle in a single template duplex, there are two copies of the template duplex consisting of one new and one old DNA strand • With each cycle, the newly synthesized DNA serves as templates in later cycles • After numerous cycles of PCR, the target sequence will be exponentially amplified millions of times UNIT II: VISUALIZING DNA ON A GEL GEL ELECTROPHORESIS • Allows visualization of DNA molecules • General technique that can be utilized to separate DNA fragments, and other macromolecules (i.e. RNA and proteins) from sources such as in PCR • Based on rate of movement through an agarose gel in an electric field PROCESS OF GEL ELECTROPHORESIS • During gel electrophoresis, molecules are loaded into wells of porous gel, before traveling through its length due to an electrical field • A positive charge is established at one end of the gel, and a negative charge at the other • Since DNA/RNA are negatively charged due to ionized phosphate groups, they will be attracted towards the positively charged/anode end • Molecules that travel through the pores at the highest speed tend to be smallest, while the largest travel slower and therefore smaller distances • Bands are typically not visible while separating (although shown in the figure below) DNA FRAGMENT MIGRATION • Molecule separation ranges from 100s to 10,000 of nucleotides • Results are typically amplification of a single size of DNA, as well as visualization of a sample containing a mixture of DNA fragments of various sizes • A standardized ladder (containing DNA fragments of known sizes) is also loaded onto the gel simultaneous to the other samples • After the process, fragments are visualized using ultraviolet fluorescence dyes that intercalate/stain the DNA with various bands UNIT III: DNA SEQUENCING WHOLE GENOME SEQUENCING (1975) • Method developed by Frederick Sanger which is able to determine the sequence of a DNA molecule • Limitation was that it could only determine the sequence of small DNA fragments SHOTGUN SEQUENCING • Gene Myers and Jim Weber invented an approach for large-scale sequencing projects and facilitated whole genome DNA sequencing • Ability to break the entire genome into different sized pieces, and then proceeding with three specific phases PHASE I: random DNA sequencing in each fragment PHASE II: identification of overlapping regions between generated fragments, and inferring/assembling of the sequence of nucleotides in the DNA composing each chromosome PHASE III: annotating the sequences to best identify the gene-encoding, regulatory, and non-coding regions TIMELINE OF GENOMIC SEQUENCING 1995: first whole genome to be sequenced, Haemophilus influenza 1998: first multicellular organism genome to be sequenced, C. elegans 1999: sequencing of the Drosophila 2000: entire human genome sequenced SANGER SEQUENCING/DIDEOXY CHAIN-TERMINATION METHOD • DNA to be sequenced serves as a template for DNA synth
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