Proceedings of the National Academy

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• Proceedings of the National Academy
• September 9, 2002

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Virus detection is TRICKY!

• large number of viral serotypes,
• serotype same virus, different antisera
  specificity,
• infections involving multiple viruses,
• high homology between relatives.

3
Old Techniques

• required isolation and in vitro cultures and
  immunological assays.

Shell vials,

• direct fluorescence antibody,
• enzyme immunoassay.

4
PCR Revolutionized Virus Identification

• facilitates detection of several viruses in
  parallel,
• through multiplexing of species specific primers,
  

5
The Idea

• Make a DNA microarray from representative
  sequences from multiple viral families!

• The design
• 70-nt oligonucleotides based on pre-existing
• viral genome sequences,
• forward and reverse sequences used,
• human sequences as controls.

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And this helps how??

• one test, multiple (100s) viruses detectable,
• only small amounts of sample required (no
  culture),
• sensitive detection between relatives,
• less labor and time intensive,
• not testing for a specific candidate or family,
  so no inherent bias,
• able to detect unknown viruses.

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The Wonders of Technology

• BLAST/N (free Bioinformation),
• establishes nucleotide similarities in/between
  families,
• RNeasy,
• kit for facile isolation of RNA from cells,
• Random PCR,
• amplifies sample target DNA without bias,
• HeLa Cells,
• immotalized human cells.

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Making the Arraychoosing 70-mers Targets

• Download virus sequences from the free public
  database,
• Divide each into 70 base pair portions,
• compare these portions with the entire viral
  sequence database, looking for highly conserved
  regions.

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Basic Local Alignment Search ToolBlast

• sequence similarity finder,
• identifies sequence alignments between a sequence
  of interest, and a database of sequences,
• exact matches are scored and are weighed against
  single base mismatches, gaps and deletions,
• a score is obtained that provides some measure of
  the sequences similarity.

BlastN compares a DNA sequence with a nucleotide
database.
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TTAAAACAGCTCTGGGGTTGCTCCCACCCCAGAGGCCCACGTGGCGGCTA
GTAATCTGGTATCAGGTACCTTTGTACGCCTGTTTTATATCCCTTCCCCC
GTAACTTTAGAAGCTTATCAAAAGTTCAATAGCAGGGGTACAAGCCAGTA
CCTCTACGAACAAGCACTTCTGTTTCCCCGGTGAAATCATATAGACTGTA
CCCACGGTCAAAAGTGATTGATCCGTTATCCGCTTGAGTACTTCGAGAAG
CCTAGTATCGCCTTGGAATCTTCGACGCGTTGCGCTCAACACTCTGCCCC
GAGTGTAGCTTAGGCTGATGAGTCTGGGCACTCCCCACCGGCGACGGTGG
CCCAGGCTGCGTTGGCGGCCTACCCATGGCTGATGCCGTGGGACGCTAGT
TGTGAACAAGGTGTGAAGAGCCTATTGAGCTACTCAAGAGTCCTCCGGCC
CCTGAATGCGGCTAATCCTAACCACGGAGCAATCGCTCACGACCCAGTGA
GTAGGTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTG
TCCGTGTTTCCCTTTATATTCATACTGGCTGCTTATGGTGACAATTTACG
AATTGTTACCATATAGCTATTGGATTGGCCACCCAGTGCTGTGCAATATA
TTTGAGTGCTTCTTTCATAGGTGTTACCAACATCACATTTAAACCACAAT
AGTCAGTGCAAATGGGGGCTCAAGTTTCAACCCAAAAGACCGGTGCACAC
GAGAATCAAAACGTGGCAGCCAATGGATCCACCATTAATTATACTACCAT
CAACTACTACAAAGACAGCGCGAGTAACTCCGCTACTAGACAAGACCTCT
CCCAAGATCCATCAAAATTCACAGAACCGGTTAAGGACTTAATGTTGAAA
ACAGCACCAGCTTTAAACTCGCCCAACGTGGAAGCATGTGGGTACAGTGA
CCGTGTGAGGCAAATCACTTTAGGTAACTCGACCATTACCACACAGGAAG
CAGCTAATGCTATTGTTGCTTATGGTGAGTGGCCCACTTACATAAATGAC
TCAGAAGCTAATCCGGTAGATGCACCCACTGAACCAGACGTTAGTAGCAA
CCGTTTTTACACCCTGGAATCGGTGTCTTGGAAGACCACTTCAAGGGGAT
GGTGGTGGAAGCTACCAGATTGTCTAAAAGATATGGGAATGTTTGGTCAG
AATATGTACTATCACTACTTAGGACGCTCTGGTTACACCATTCATGTCCA
GTGCAACGCTTCTAAGTTTCACCAAGGGGCGTTAGGAGTTTTCCTGATAC
CAGAGTTTGTCATGGCTTGCAACACTGAGAGTAAAACATCATATGTTTCA
TACATCAACGCAAATCCTGGTGAAAGGGGCGGTGAGTTCACGAACACCTA
CAACCCATCAAACACTGATGTCAGTGAGGGCAGACAGTTCGCAGCACTGG
ATTATCTGCTGGGTTCTGGTGTCCTAGCAGGAAACGCTTTCGTATACCCG
CACCAGATCATCAATTTGCGCACCAACAACAGTGCAACAATTGTGGTACC
ATATGTGAACTCGCTCGTGATTGATTGTATGGCAAAACACAATAACTGGG
GTATTGTCATATTACCACTGGCACCCTTGGCCTTTGCCGCAACATCGTCA
CCACAGGTGCCTATTACAGTGACCATCGCACCCATGTGTACAGAATTTAA
TGGGTTGAGAAATATTACCATCCCAGTACATCAAGGGTTGCCAACAATGA
ACACACCTGGTTCCAATCAATTCCTTACATCTGATGACTTCCAGTCGCCC
TGTGCCTTACCTAATTTTGATGTTACTCCACCGATACACATACCCGGGGA
AGTGAAGAATATGATGGAACTAGCTGAAATTGACACACTGATCCCAATGA
ACGCAGTGGACGGGAAGGTGAACACAATGGAAATGTATCAAATACCATTG
AATGACAATTTGAGCAAGGCACCTATATTCTGTCTATCTTTATCACCTGC
TTCTGACAAACGACTGAGTCACACCATGTTGGGTGAAATTCTAAATTATT
ACACTCATTGGACGGGGTCCATCAGGTTCACCTTTCTATTTTGTGGTAGT
ATGATGGCCACTGGTAAACTGCTCCTCAGCTATTCCCCACCAGGAGCTAA
ACCACCAACCAATCGCAAGGATGCAATGCTAGGTACACACATCATCTGGG
ACCTAGGGTTACAATCCAGTTGTTCCATGGTTGCACCGTGGATCTCTAAC
ACAGTGTACAGGCGGTGTGCACGTGATGACTTCACTGAGGGCGGGTTTAT
AACTTGCTTTTATCAAACTAGAATTGTTGTGCCTGCTTCAACCCCCACCA
GTATGTTCATGTTAGGCTTTGTTAGTGCGTGTCCAGATTTCAGTGTCAGA
CTGCTTAGGGACACTTCCCATATTAGTCAATCAAAACTAATAGCACGCAC
ACAAGGCATTGAAGACCTCATTGACACAGCGATAAAGAATGCCTTAAGAG
TGTCCCAACCACTTCGGCCCAGTCAACTGAAGCAACCCAATGGAGTAAAT
AGCCAGGAGGTGCCAGCTCTAACTGCTGTGGAAACAGGAGCATCTGGTCA
AGCGATCCCCAGTGATGTGGTGGAAACTAGACACGTGATAAATTACAAAA
CCAGGTCTGAATCGTGTCTTGAGTCATTCTTTGGGAGAGCTGCGTGTGTC
ACAATCCTATCTCTGACCAACTCTTCCAAGAGCGGGGAGGAGAAGAAGCA
TTTCAACATATGGAACATTACATACACCGACACTGTTCAGTTACGCAGAA
AATTGGAGTTTTTCACGTATTCCAGGTTTGACCTTGAAATGACTTTTGTG
TTCACAGAGAATTACCCTAGTACAGCCAGTGGGGAAGTGCGTAACCAATG
TGACCAGATCATGTACATTCCACCAGGGGCACCCCGTCCATCATCCTGGG
ATGACTATACATGGCAATCCTCCTCTAACCCTTCCATCTTTTACATGTAT
GGAAATGCACCTCCACGGATGTCAATTCCTTACGTGGGGATTGCCAATGC
CTATTCACACTTCTATGATGGCTTTGCACGGGTGCCACTTGAGGGTGAGA
ATACCGATGCTGGTGACACGTTTTACGGCTTAGTGTCCATAAATGATTTT
GGAGTTTTAGCAGTTAGAGCGGTAAACCGCAGTAACCCACACACTATACA
CACATCTGTGAGAGTGTACATGAAACCAAAACACATTCGGTGTTGGTGCC
CCAGACCTCCTCGGGCTGTATTATACAGGGGAGAGGGAGTGGACATGATA
TCCAGTGCAATTCAACCTCTGACTAAAGTAGACTCAATTACCACTTTTGG
GTTTGGCCACCAGAACAAGGCAGTGTACGTTGCCGGTTACAAGATTTGTA
ACTACCACCTAGCAACCCCAAGTGATCACTTGAATGCAATTAGTGTGTTA
TGGGACAGGGATTTAATGGTGGTGGAATCTAGAGCCCAGGGAACTGATAC
CATTGCCAGGTGTAGTTGCAGGTGTGGAGTTTACTATTGTGAATCTAGAA
GGAAGTACTACCTTGTCACTTTCACTGGCCCAACGTTTCGATTTATGGAA
GCAAATGACTACTATCCAGCAAGATACCAGTCTCACATGCTGATAGGGTG
CGGATTTGCAGAACCCGGGGACTGTGGTGGGATATTAAGGTGCACTCATG
GGGTGATTGGTATCATCACTGCAGGGGGTGAAGGGATAGTAGCCTTTGCT
GACATTAGAGACCTTTGGGTGTATGAAGAGGAAGCCATGGAACAGGGAAT
AACAAGTTACATCGAATCCCTCGGTACAGCTTTTGGTGCAGGGTTCACCC
ACACAATCAGTGAGAAAGTGACTGAGTTGACAACAATGGTCACCAGTACT
ATCACAGAAAAATTACTGAAAAATTTAGTGAAAATAGTATCGGCTCTAGT
GATTGTTGTGAGAAATTATGAGGACACTACCACGATCCTTGCAACACTAG
CATTACTTGGGTGTGATATATCTCCTTGGCAATGGTTGAAGAAGAAGGCA
TGTGACTTACTAGAGATTCCTCATGTGATGCGCCAAGGTGATGGGTGGAT
GAAGAAATTCACAGAGGCTTGCAATGCAGCTAAAGGGCTTAGATGGGTCA
GCAATAAAATTTCCAAGTTTGTAGATTGGTTGAAGTGTAAAATTATCCCA
GAAGCTAAGGACAAGGTGGAATTTCTCACCAAGTTGAAACAGCTAGACAT
GCTGGAAAATCAAATTGCAACCATCCACCAATCTTGCCCCAGCCAAGAAC
AACAAGAGATCCTTTTCAACAACGTAAGATGGCTAGCAGTCCAGTCCCGT
CGGTTCGCGCCATTATATGCTGTGGAGGCACGCCGGATTAGCAAAATGGA
GAGCACAATAAACAATTATATACAGTTCAAGAGCAAACACCGTATTGAGC
CAGTATGTATGCTCGTTCATGGGTCACCAGGGACGGGTAAGGGTATAGCT
AGCTCATTGATAGGTAGAGCAATAGCAGAGAGGGAAACCACATCGGTCTA
CTCAGTGCCACTGGCCCCATCTCACTTTGATGGCTATAAACAACAAGGGT
ATGATATGGACGACCTAAACCAAAACCCCGATGGTATGGACATGAAACTG
TTTTGCCAAATGGTATCAACAGTGGAGTTCATTCCTCCAATGGCCTCACT
AGAGGAGAAGGGTATTTTGTTTACATCTGATTATGTCCTGGCTTCTACCA
ACTCTCACTCAATAGCACCACCCACAGTAGCTCATAGTGATGCCTTAACC
AGACGATTTGCATTTGATGTGGAGGTTTACACGATGTCTGAACATTCAGT
CAAAGGCAAACTAAACATGGCCACAGCCACTCAGTTGTGTAAGGATTGTC
CAACACCTGCAAATTTCAAAAAGTGTTGCCCTCTCGTCTGTGGAAAGGCC
TTGCAATTAATGGACAGGTACACCAGACAGAGGTTCACTGTGGATGAGAT
TACCACATTAATCATGAATGAGAAAAACAGAAGGGCCAACATTGGCAATT
GTATGGAAGCCTTGTTTCAAGGACCACTGAGGTACAAAGATCTGAAGATT
GATGTGAAGACAGTTCCCCCCCCTGAGTGCATCAGTGATTTATTACAAGC
AGTGGATTCTCAAGAGGTTAGGGATTACTGTGAGAAGAAAGGCTGGATCG
TTAACGTTACTAGCCAGATACAATTAGAAAGGAACATCAATAGGGCCATG
ACTATACTCCAAGCTGTTACCACATTTGCAGCAGTCGCAGGAGTAGTGTA
TGTAATGTACAAACTCTTCGCTGGCCAGCAGGGTGCATACACTGGCTTGC
CAAACAAAAAGCCCAATGTCCCTACTATCAGAATTGCTAAAGTCCAGGGG
CCAGGATTTGACTATGCAGTGGCAATGGCAAAAAGAAACATAGTTACTGC
AACCACCACTAAGGGTGAATTTACCATGTTAGGGGTGCATGATAATGTAG
CAATATTGCCAACCCATGCCGCTCCAGGAGAAACTATCATTGTTGATGGG
AAAGAAGTGGAGATCCTAGATGCCAGAGCCTTAGAAGATCAAGCGGGAAC
CAACCTTGAGATTACCATTATTACTCTAAAAAGAAATGAGAAATTTAGAG
ACATCAGACCACATATTCCCACCCAAATTACTGAAACTAACGATGGAGTG
TTGATCGTGAACACTAGTAAGTACCCCAACATGTATGTCCCAGTTGGTGC
TGTGACTGAACAGGGATATCTTAATCTCAGTGGACGCCAAACTGCTCGTA
CTTTAATGTACAACTTTCCAACACGAGCAGGCCAGTGCGGAGGAATCATC
ACTTGTACTGGCAAAGTCATTGGGATGCACGTTGGCGGGAACGGTTCACA
TGGGTTTGCAGCAGCCCTTAAGCGATCATACTTCACCCAAAATCAGGGCG
AAATTCAATGGATGAGGTCATCAAAAGAAGTGGGGTACCCCATTATAAAT
GCCCCATCCAAGACAAAGTTAGAACCCAGTGCTTTTCACTATGTTTTTGA
AGGTGTTAAGGAACCAGCTGTGCTCACTAAAAATGACCCCAGACTAAAAA
CAGATTTTGAAGAAGCCATCTTTTCAAAATACGTGGGGAACAAAATTACT
GAAGTGGACGAATACATGAAAGAAGCAGTGGATCACTATGCAGGACAGTT
AATGTCACTGGATATCAACACAGAACAGATGTGCCTGGAGGATGCCATGT
ACGGTACTGATGGTCTTGAAGCTCTGGATCTTAGCACTAGTGCTGGATAC
CCTTATGTTGCAATGGGGAAAAAGAAAAGAGACATTCTAGACAAACAGAC
CAGGGATACTAAAGAGATGCAGAGACTTTTGGACACCTATGGAATCAACC
TACCATTAGTCACATACGTGAAAGATGAACTCAGATCAAAGACTAAAGTG
GAACAAGGAAAGTCAAGGTTGATTGAAGCTTCCAGTCTTAATGATTCAGT
TGCAATGAGAATGGCCTTTGGCAATCTTTACGCAGCTTTCCACAAGAATC
CAGGCGTGGTGACAGGATCAGCAGTTGGTTGTGACCCAGATTTGTTTTGG
AGCAAGATACCAGTGCTAATGGAAGAAAAACTCTTCGCTTTTGACTACAC
AGGGTATGATGCCTCACTCAGCCCTGCTTGGTTTGAAGCTCTTAAAATGG
TGTTAGAAAAGATTGGATTCGGCAATAGAGTAGACTACATAGACTACCTG
AACCACTCTCACCACCTTTATAAAAACAAAACTTATTGTGTCAAAGGTGG
CATGCCATCCGGCTGCTCTGGCACATCAATTTTCAACTCAATGATTAATA
ACCTGATCATTAGGACGCTTTTACTGAGAACCTACAAGGGCATAGACTTG
GACCACCTAAAAATGATTGCCTATGGTGATGACGTGATAGCTTCCTACCC
CCATGAGGTTGACGCTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGAC
TGACCATGACTCCAGCAGATAAATCAGCAACCTTTGAAACAGTCACATGG
GAGAATGTAACATTCCTGAAAAGATTTTTCAGAGCAGATGAGAAGTATCC
ATTCCTGGTGCATCCAGTGATGCCAATGAAAGAAATTCACGAATCAATCA
GATGGACCAAGGACCCTAGAAACACACAGGATCACGTACGCTCATTGTGC
CTATTGGCTTGGCATAACGGCGAAGAAGAATATAATAAATTTTTAGCTAA
AATCAGAAGTGTGCCAATCGGAAGAGCTTTATTGCTCCCAGAGTACTCCA
CATTGTACCGCCGATGGCTCGATTCATTTTAGTAACCCTACCTCAGTCGG
ATTCGGATTGGGTTATACTGTTGTAGGGGTAAATTTTTCTTTAATTCGGA
G
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gt 20 Nucleotide Matches Significant
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Spotting the Probes

• For most of the viral sequences, the five best
  70-mers were chosen,
• plus the anti-sense sequence (why?),
• These were spotted onto the solid substrate.

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HeLa Cells?targets

• Cervical cancer cells from Henrietta Lacks, 1951,
• Cultured and used worldwide for study.

• In this study
• Infected cell cultures with viruses of interest
  (KSHV aka HHV-8, Poliovirus1, rhinoviruses,
  enteroviruses, hepatitis B and C,
  papillomavirus),
• Then infected people with virus,
• simple colds (OK work if you can get it).

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Making the Targets

• Collect nasal lavage (snot),
• or cell cultures,
• Extract RNA,
• reverse transcriptase with degenerate primers,

5 - GTTTCCCAGTAGTCTCNNNNNNNN - 3
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Degeneracyprimers

• 5 - GTTTCCCAGTAGTCTCNNNNNNN - 3
• the N(s) can be any base, thus all of the 65,536
  possible combinations are made,
• random primes bind to complementary sequences,
• reverse transcriptase builds off the 3 end,
• some of the primers face each other,
• PCR is then performed using...
• 5 - GTTTCCCAGTAGTCTC - 3

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Labeling Targets

• Cy3 (uninfected)
• green
• people,
• cell lines,
• Fluorescent base inserted into the sequence.

• Cy5 (infected)
• red
• people,
• cell lines,
• Fluorescent base inserted into the sequence.

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Summary(so far)
Extract RNA
RT/PCR
Label
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SO NEATO!
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Lets get people sick!
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Snot from off the street
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At least they didnt have canine distemper or
bovine parainfluenza!
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Such a diagnostic tool will undoubtedly have
many uses in the study of viral pathogenesisas
well as in instances of BIOTERRORISM.
(send money)
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Quiz Questions!

• What are some of the potential uses of this
  technology?
• How does this improve on old techniques?

Hmmm
Ponder, Ponder