DNA Technology

  • Polymerase chain reaction (PCR) -> repeated 30-40 cycles 

    • Problem: not enough DNA to run many tests so PCR produces billions of copies in short period of time, helps study DNA/read small samples
    • Components: dNTPS(DNA nucleotides/deoxyribonucleotides), primers (binds to gene and add to 3’ side, short 15-20 bp DNA fragments with specific seq), Taq polymerase (DNA poly from thermus aquaticus, bacteria from hot spring)

  • Denaturation: H-bonds break at high temperatures ( >90 C), separates DNA strands

  • Annealing: primer binds to target, 2 DNA primers used, short synthetic strands (parent: 3’ -> 5’; daughter: 5’ -> 3’), 40C - 65C (cooling), primers anneal to 3’ side of DNA

  • Extension: builds new DNA fragments, 72C (Taq poly replicates DNA strand), 5’ -> 3’ (both daughter strands are identical copies), at end of 1 cycle -> 2 new DNA strands

    • After 3, most strands are target sequence, after many can be 10^6/10^9 increase in DNA 

  • Gel electrophoresis

    • Polyacrylamide gel (agarose polysaccharide), separates by size, DNA fragments go from - to +, smaller ones move far and larger ones stay close, used in conjunction with other mol. Techniques

  • DNA sequencing 

    • Sequence: figure out nucleotide order of 1 gene, uses PCR/electrophoresis -> dideoxy chain termination (DCT/Sanger method)
    • Dideoxy chain termination method (dideoxyribonucleic acid); no free OH and strand can’t elongate
    • Ingredients: template DNA to be sequenced (know length but not base order), primers (short DNA segments, starter), many of all 4 types of deoxyribonucleotides(normal DNA), DNA poly, key to sequencing: few of each 4 dideoxyribonucleotides (labeled with fluorescent mol. ddNTPs of last nucleotide inserted)

  • Process of DNA sequencing 

    • Thermocycler denatures DNA fragment, anneal primer to template, elongate: DNA poly adds NTPs
    • As reaction proceeds results in many strands, sort by length in capillary gel electrophoresis, sequence revealed (complementary to template strand), spectrogram results are graph

  • Uses for sequencing: explore evolutionary relationships, discover sequence of DNA fragment, determine loci/function

    • Application of DNA tools 

  • Species diversity: telling if 2 types of algae are same species

    • Used to identify variety of different species comprising community, good for rare/highly mobile/difficult to ID/microscopic

  • Bar coding: used to ID species, short seq. From standard part of genome

    • Among all members of large group but varies between species, Ex: 648 bp region in mitochondrial cytochrome C oxidase 1 gene 

  • Gene therapy: treatment for disease (recently FDA-approved), CRISPR: silences/inserts gene into living cells 

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