The Genomicist's Tool Kit:

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The Genomicist's Tool Kit
DNA Cloning and Sequencing Polymerase Chain
Reaction, DNA Microarrays
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The library
Copying
Export
Translation
Genetic information is stored in DNA
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Sugar-phosphate backbone
Base pair
Nitrogenous base
The DOUBLE HELIX DNA
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DNA strands stick together
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When DNA replicates, the strands separate
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Each strand is copied precisely
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DNA is stored in the nucleus
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Decoding DNA
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Each codon places a different amino acid in the
protein.
The genetic code
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Proteins provide structure
Proteins do work
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How do we decode the information in DNA?

• Cloning DNA means making many
• copies of small sections of the DNA

• Sequencing DNA means determining
• the exact order of the nucleotides

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Plasmids are tiny circular chromosomes in
bacteria
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Splicing DNA with restriction enzymes
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EcoRI is a restriction enzyme
DNA ligase covalently bonds the recombinant DNA
molecule
Making a recombinant plasmid
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Making many copies of the recombinant DNA
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The primer is synthesized chemically A primer
is an oligonucleotide
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AGTACGTAGTGATGCAAAGGACA TGCATCACT
AGTACGTAGTGATGCAAAGGACA TGCATCACTA
nucleotides DNA polymerase
AGTACGTAGTGATGCAAAGGACA TGCATCACTAC
AGTACGTAGTGATGCAAAGGACA TGCATCACTACG
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGT
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTT
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTTT
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTTTC
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTTTCG
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTTTCGA
AGTACGTAGTGATGCAAAGGACA TGCATCACTACGTTTCGAG
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T
A
C
G
T
T
T
C
G
A
G
G A T C
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Shortest Fragments separated by length
Longest
A DNA sequencing gel
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A contemporary ABI sequencing machine
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PCR the polymerase chain reaction
Polymerase chain reaction or PCR
1. Denature DNA 2. Anneal primers 3. Extend
with Taq DNA polymerase Repeat 25-40
times, Sequence if desired
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How do PCR and DNA sequencing affect me?

• You have a genetic fingerprint
• Establishing paternity
• Diagnosing genetic diseases
• Forensic diagnostics

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You are genetically unique
The DNA of any two individuals differs by about 1
nucleotide in a thousand this is a SNP or single
nucleotide polymorphism
The differences can be just one nucleotide
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Length polymorphisms (differences)
Some genes have many copies of short repeats of
2, 3, or 4 nucleotides
PCR amplified fragments from variable-length
genes can be separated by size
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DNA chips or microarrays what are they?

• DNA chips are arrays of DNAs PCR amplified
  from genes
• DNA microarrays are also made using
  oligonucleotides
• Microarrays are used to analyze gene
  expression levels
• Gene expression is determined from RNA levels

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Gene are expressed in mRNAs (transcript)
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Gene 1
Gene 2
Gene 3
Gene 3 is transcribed infrequently
Gene 2 is moderately transcribed
Gene 2 is abundantly transcribed
Genes are transcribed at very different
frequencies
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Complementary strands of DNA or RNA anneal very
precisely.
Nucleic acid hybridization
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(No Transcript)
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The Penn State microarray robot
John Szot Director PSU Microarray Facility
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G
Gene X DNA
Hybridize to DNA
Hybridized
Extract RNA
View at wavelength 1
View at wavelength 2
Transcripts hybridize in proportion to their
numbers
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Microarray experiments describe how gene
expression changes in time
From Iyer et al., Science 283 83 (1999)
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What questions can we ask using microarrays?

• How are gene networks organized?
• Are there particular genes work (or not
  working) in
• each kind of cancer?
• Whats the gene expression profile of a
  disease?
• What are the genes profile of the fittest?

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Sources of illustrations http//www.accessexce
llence.org Lectures by S. Cohen and E.
Landers Graphics Gallery
http//www.bis.med.jhmi.edu/Dan/DOE/intro.html Pr
imer on Molecular Genetics http//www.appliedbi
osystems.com http//www.phage.org/links_dna_seq
uencing.htm