DNA Sequencing

Taste Test How can you run multiple tests on a small sample size? When only have a small sample size and need to run multiple tests, create more sample size!

Polymerase Chain Reaction Replicate DNA regions Quick process Occurs within lab No nucleus required 3 main steps Denaturation Annealing Elongation PCR produces copies of DNA in a short amount of time Replicate regions of DNA, not entire genome Process does not require nucleus (no cell required)

When will you have only target DNA? Step 1 – denaturation DNA molecule is denatured and separated by heat Breaks hydrogen bonds holding strands together Produce single-stranded DNA Step 2 – annealing Occurs at lower temperature DNA primers join single-stranded DNA at 3’ end allows orientation of DNA synthesis Step 3 – elongation DNA polymerase binds to primer Specific DNA polymerase known as Taq polymerase is used Can withstand high temperatures Synthesizes new DNA strand Newly synthesized DNA strands are slightly shorter than original No complete strands until third cycle because of primers Process is repeated for another cycle to produce multiple copies Will produce 2n copies, where n is the number of cycles How many in 5 cycles? When will you have only target DNA?

Gel Electrophoresis Chromatography-like Uses chemical and physical properties of DNA Separates fragments of DNA Negative and positive terminals

DNA sample with loading dye is placed in agarose gel wells Molecular marker is loaded into last well as reference of fragments Molecular marker contains known sizes of DNA fragments Used as reference Buffer is added above gel Buffer is required for movement of DNA fragments and Negative and positive terminals are attached and machine is turned on DNA will separate because negatively charged Will move away from negative terminal and towards positive terminal Lighter more positive fragments will travel further Gel is stained with ethidium bromide to see fragments Need to stain because otherwise won’t be able to see small fragments UV light is used to see glow from ethidium bromide

Chain Termination Method Frederick Sanger Sometimes referred to as Sanger method Labelled dideoxynucleotide (ddNTP) Used as dyes Stops elongation process Fluoresce under laser light

Sanger Method Occurs in reaction tubes Each tube will contain ddNTP, DNA, DNA polymerase, and a DNA primer DNA to be sequenced will bind with primer Polymerase will put nucleotides to build new strand When ddNTP binds to new strand, synthesis will stop Gel electrophoresis to see seqeunce Sanger Method

Whole-Genome Shotgun Method Craig Venter Faster and longer sequences DNA randomly cut Passed through pressurized syringe Cloned in plasmids Sequenced using Sanger method Computer analyzes partial sequences

What is one major drawback? DNA broken into smaller fragments Sequenced by computer analysis Match overlapping sequences Can be problematic if repeating sequence Near impossible to match because don’t know how long repeats are Shotgun Sequencing What is one major drawback?

Bioinformatics Analyze sequences Structural genomics Locate genes within genome Functional genomics Role proteins play Nanopore sequencing Tiny sub-microscopic holes Microarray Use probe to locate gene Bioinformatics refer to the use of computer technology to process biological data Structural genomics is the study of the structure of genes and their locations in a genome; analysis of the nucleotide sequences to locate genes within the genome Functional genomics is the study of the functions of genes, the proteins they make, and how these proteins function Nanopore sequencing pulls DNA through a small hole Hole is so small that only one strand of DNA will fit through Current passed through sheet Computer analyzes current passed through each base pair

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