Sequencing DNA and the Polymerase Chain Reaction

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Sequencing DNA and the Polymerase Chain Reaction

• Core 218
• Biotechnology and Society
• Spring 2007

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DNA Sequencing

• DNA sequencing is the determination of the
  precise sequence of nucleotides in a sample of
  DNA
• There are two methods that were commonly used to
  sequence DNA.
• Sanger Method dideoxy method
• Maxam-Gilbert Method chemical degradation method

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Genomics

• Investigations into the structure and function of
  very large numbers of genes, undertaken in a
  simultaneous fashion.
• Structural Genomics - the effort to determine 3D
  structures of proteins from experimentation and
  computer analysis
• Functional Genomics determine the function of
  genes by determining their role in a model
  organism.

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Developments in Functional Genomics

• Complete the sequence of the roundworm C. elegans
  genome by 1998.
• Complete the sequence of the fruit fly Drosophila
  genome in 2002.
• Develop an integrated physical and genetic map
  for the mouse, generate additional mouse cDNA
  resources, and complete the sequence of the mouse
  genome by 2008.
• Identify other useful model organisms and support
  appropriate genomic studies.

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Functional Genomics

• Full-length cDNA clones and sequences that
  represent human genes and model organisms.
• Functions of nonprotein-coding sequences.
• Develop technology for comprehensive analysis of
  gene expression.
• Improve methods for genome-wide mutagenesis.
• Develop technology for large-scale protein
  analyses.

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Genomics

• Bioinformatics
• Improve content and utility of databases.
• Tools for data generation, capture, and
  annotation.
• Tools and databases for comprehensive functional
  studies.
• Tools for representing and analyzing sequence
  similarity and variation.
• Exportable software that can be widely shared.

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Genome, Chromosome

• Definitions
• Genome - All the DNA contained in an organism or
  a cell, which includes both the chromosomes
  within the nucleus and the DNA in mitochondria.
• Chromosome - One of the threadlike "packages" of
  genes and other DNA in the nucleus of a cell.
  Different kinds of organisms have different
  numbers of chromosomes. Humans have 23 pairs of
  chromosomes. Each parent contributes one
  chromosome to each pair, so children get half of
  their chromosomes from their mothers and half
  from their fathers.

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Gene, Nucleotide

• Definitions
• Gene - The functional and physical unit of
  heredity passed from parent to offspring. Genes
  are pieces of DNA, and most genes contain the
  information for making a specific protein.
• Nucleotide - One of the structural components, or
  building blocks, of DNA and RNA. A nucleotide
  consists of a base (one of four chemicals
  adenine, thymine, guanine, and cytosine) plus a
  molecule of sugar and one of phosphoric acid.

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Genome Size Examples
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The Importance of Sequence Information

• Paul Berg All human disease is genetic in
  origin (discussion)
• James Watson What we need to do now is to find
  those genes
• Ex. Huntingtons Disease
• Currently no cure
• Can test genetically would you do it?

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The Importance of Sequence Information

• Genetic Disease Predisposition
• Over 4,000 known genetic disorders
• More where genetic malfunctions play a role in
  disease
• New drug development
• DNA information for natural pharmaceuticals
• Ex. plasminogen activator, interferon, and
  erythropoietin
• Classification (taxonomy)

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In the early 60s

• Yeast alanine tRNA (75-80 nucleotides)
• Holley, Cornell, enzymes, specific fragments,
  degradation
• 1975, Walter Fier, complete genome of a single
  stranded RNA phage

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By the 1970s

• We had a lot of enzymes
• Lots of fragments
• No way to determine sequence
• Except with small RNA fragments
• Therefore, the hard way cut DNA into small
  pieces, use RNA polymerase, sequence RNA fragment

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Sequence Masters

• Fred Sanger, 1958
• Was originally a protein chemist
• Made his first mark in sequencing proteins
• Made his second mark in sequencing RNA
• 1980 dideoxy sequencing

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The Sanger Method

• Random incorporation of a dideoxynucleoside
  triphosphate into a growing strand of DNA
• Requires DNA polymerase I.. Why?
• Requires a cloning vector with initial primer
  (M13, high yield bacteriophage, modified by
  adding beta-galactosidase screening,
  polylinker)
• Uses 32P-deoxynucleoside triphosphates

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Sanger Method Sequencing Gel
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Sequence Masters

• Walter Gilbert
• Harvard physicist
• Knew James Watson
• Became intrigued with the biological side
• Became a biophysicist
• Allan Maxam

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The Maxam-Gilbert Technique

• Principle - Chemical Degradation of Purines
• Purines (A, G) damaged by dimethylsulfate
• Methylation of base
• Heat releases base
• Alkali cleaves G
• Dilute acid cleave AgtG

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The Maxam-Gilbert Technique

• Principle Chemical Degradation of Pyrimidines
• Pyrimidines (C, T) are damaged by hydrazine
• Piperidine cleaves the backbone
• 2 M NaCl inhibits the reaction with T

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The Maxam-Gilbert Method
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Comparison

• Sanger Method
• Enzymatic
• Requires DNA synthesis
• Termination of chain elongation

• Maxam Gilbert Method
• Chemical
• Requires DNA
• Requires long stretches of DNA
• Breaks DNA at different nucleotides

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Sequencing Gives

• The letters in a sentence
• Remember Prions?
• Short sequence in genomes
• Single nucleotide change in alleles
• Valine - Valine not susceptible to BSE
• Methionine - Valine at risk
• Methionine-methionine watch out!
• How can we genetically screen for single
  nucleotide differences?

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Answer PCR

• PCR Polymerase Chain Reaction
• Requires
• Polymerase from Thermus aquaticus
• Hi and low temperature reactions
• Template DNA
• Primer
• Nucleosides (A, T, G, C)

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PCR

• PCR Polymerase Chain Reaction, a method that
  uses a high temperature DNA polymerase, DNA
  separation and reannealing, and repetitive
  cycles, to amplify fragments of DNA in vitro.

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Importance of PCR

• Allowed rapid advances in genomics
• Enabled detection of single genes
• Forensics
• Sequencing

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Tutorials In Molecular Biology
Dr. Lianna Johnson
An excellent site by
Design and Programming
Steve Sobolevsky
John Bacheller, Grant Alkin and Eric Simkin
Contributions
Developed in part with support from a Howard
Hughes Medical Institute grant through the
Undergraduate Biological Sciences Education
Program to the University of California, Los
Angeles.
http//www.lsic.ucla.edu/ls3/tutorials/index_menu.
html