Transcript Automated Searching of Polynucleotide Sequences

Automated Searching of
Polynucleotide Sequences
Michael P. Woodward
Supervisory Patent Examiner - Art Unit 1631
571 272 0722
[email protected]
John L. LeGuyader
Supervisory Patent Examiner - Art Unit 1635
571 272 0760
[email protected]
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Standard Databases
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GenEMBL
N_Genseq
Issued_Patents_NA
EST
Published_Applications_NA
.rge
.rng
.rni
.rst
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Databases at Time of
Allowability
• Pending_Patents_NA_Main
• Pending_Patents_NA_New
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.rnpn
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Types of Nucleotide Sequence Searching
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Standard (cDNA)
Oligomer
Length Limited Oligomer
Score over Length
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Types of Nucleotide Sequence Searching
• Standard (cDNA)
– useful for finding full length hits
– the query sequence is typically the full length of
the SEQ ID NO:
– the search parameters are the default parametersGap Opening Penalty & Gap Extension Penalty of
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– standard suite of NA databases are searched
– normally 45 results and the top fifteen alignments
are provided, however, additional results and
alignments can be provided.
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Standard (cDNA) search
• Fragments and genomic sequences
are often difficult to find
• Fragments are buried in the hit list
• The presence of introns in the
database sequence results in low
scores.
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Types of Nucleotide Sequence Searching
• Standard Oligomer
– finds longest matching hits
– mismatches not tolerated in region of hit
match
• Length Limited Oligomer
– returns database hits within length range
requested
– mismatches not tolerated in region of hit
match
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Standard Oligomer Searching
• Only provides the longest oligomer
present in the sequence
• A thorough search of fragments requires
multiple searches
• Can be an effective way of finding
genomic sequences
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Standard Oligomer Searching
• the search parameters are the default
parameters-Gap Opening Penalty &
Gap Extension Penalty of 60mismatches not tolerated
• Consequently inefficient means of
finding small sequences, and with
<100% in correspondence
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Claim 1
• An isolated polynucleotide comprising
SEQ. ID. No: 1.
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Searching Claim 1
• A standard search looking for full length
hits is performed.
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Standard (cDNA) search result
0001 CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGATGG 0060
2031 CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGG---CAGATGG 2090
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Claim 2
• An isolated polynucleotide comprising at
least 15 contiguous nucleotides of SEQ.
ID. No: 1.
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Searching Claim 2
• An standard oligomer search is performed
with an oligomer length of 15 nucleotides
set as the lower limit for a hit.
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Oligomer Search Results
Standard Oligomer
CAAATGCAGGCCCCCGGACCTCCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG
Query
Database
CCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG 0060
CCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG 2500
Length Limited Oligomer
CAAATGCAGGCCCCCGGACCTCCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG
Query
Database
CCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG 0060
CCCTGCTCCTGGCTTTCGCCCTGCTCTGCCTGCCCTGG 0039
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Claim 3
• An isolated polynucleotide comprising a
polynucleotide encoding a polypeptide of
SEQ ID No: 2.
• (SEQ ID No: 2 is an Amino Acid (AA)
sequence)
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Searching Claim 3
• Seq ID No: 2 is searched against the
Polypeptide databases and it is “back
translated” and searched against the
polynucleotide databases.
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Claim 4
• An isolated polynucleotide comprising a
polynucleotide with at least 90% identity
to SEQ ID No: 1.
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Searching Claim 4
• A standard search looking for full length
hits is performed.
• Hits having at least 90% identity will
appear in the results.
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Claim 5
• An isolated polynucleotide comprising a
polynucleotide which hybridizes under
stringent conditions to SEQ ID No: 1.
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Searching Claim 5
• A standard oligomer search is performed
as well as a standard search.
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Searching Small Nucleotide
Sequences
John L. LeGuyader
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Types of Small Nucleotide
Sequences Claimed
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Fragments
Complements/Antisense
Primers/Probes
Oligonucleotides/Oligomers
Antisense/RNAi/Triplex/Ribozymes (inhibitory)
Accessible Target/Region within Nucleic Acids
Aptamers
Nucleic Acid Binding Domains
Immunostimulatory CpG Sequences
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Small Nucleotide Sequences
Claimed as Sense or Antisense?
• What is being claimed?
– Requesting the correct sequence search starts
with interpreting what is being claimed
• Complementary Sequences
– DNA to DNA: C to G
– DNA to RNA: A to U
• Matching Sequences
– A to A
– U to U
• DNA, RNA, Chimeric
• cDNA, Message (mRNA), Genomic DNA
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Impact of Sequence Identity and Length
• Size and Identity Matter
• Complements/Matches
• 100% correspondence
• Mismatches
- Varying Degrees of Percent Identity
• Gaps
- Insertion or Deletions
- Gap Extensions
• Wild Cards
• % Query Match value approximates identity
• Adjustment of search parameters (e.g.
Smith-Waterman Gap values) influences %
Query Match value
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Types of Nucleotide Sequence Searching
• Standard Search (cDNA)
• Oligomer
– finds database hits with longest regions of
matching residues
– mismatches not tolerated in region of hit match
• Length Limited Oligomer
– returns database hits within requested length
range
– mismatches not tolerated in region of hit match
• Score Over Length
– finds mismatched sequence database hits based
on requested length and identity range
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Why doesn’t a standard search of the cDNA
provide an adequate search of fragments?
• Long length sequence hits with many matches
and mismatches score higher and appear first
on the hit list, compared to short sequences
having high correspondence
– lots of regional local similarity in a long sequence
scores higher than a 10-mer with 100% identity
• Consequence
– small sequences, of 100% identity or less, are
buried tens of thousands of hits down the hit list
– most small sequence hits effectively lost
– especially for hits with <100% correspondence
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Why doesn’t a standard search of the cDNA
provide an adequate search of fragments?
• Fragments and types of sequence
searches
– Standard Search (cDNA): fragment hits
buried
– oligomer: fragment hits buried
– searching multiple fragments: millions of
hits and alignments to consider
• Each fragment of a specified sequence
and length requires a separate search
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Standard Oligomer Searching
• Won’t provide thorough search of fragments
since longer hits score higher on hit table
• Smaller size hits lost, effectively not seen
• Does not tolerate mismatches in region of
matches
• Consequently inefficient means of finding
small sequences, and with <100% in
correspondence
• Better suited to finding long sequences
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Length Limited Oligomer Searching
• Sequence request needs to set size limit
consistent with the size range being claimed
• Does not tolerate mismatches in region of
matches
• Consequently inefficient means of finding
small sequences with <100% in
correspondence
• Better suited to finding small sequences with
100% correspondence
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Score Over Length Searching
• Small oligos with <100% correspondence
– within requested length and identity (>60%) range
• Manual manipulation of first 65,000 hits
– necessitates 2+ additional hrs. of searcher’s time
– does not include computer search time
• Calculation
– Hit Score divided by Hit Length
– for first 65,000 hits of table
• Hits then sorted by Score/Length value
• First 65,000 hits likely to contain small length
sequence hits down to 60% identity
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Searching Small Sequences: Example
Consider the following claim:
• An oligonucleotide consisting of 8 to 20
nucleotides which specifically hybridizes
to a nucleic acid coding for mud loach
growth hormone (Seq. Id. No. X).
• The specification teaches that
oligonucleotides which specifically
hybridize need not have 100% sequence
correspondence.
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Mud Loach Growth Hormone cDNA
• 670 nucleotides long
• 630 nucleotides in the coding region
• 210 amino acids
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Standard Search GenBank Hit Table Against cDNA
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Standard Search GenBank Hit Table Against cDNA
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Standard Search GenBank Alignments Against cDNA
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Standard Search GenBank Alignments Against cDNA
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Oligomer Search GenBank Hit Table Against cDNA
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Oligomer Search GenBank Hit Table Against cDNA
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Oligomer Search GenBank Alignments Against cDNA
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Oligomer Search GenBank Alignments Against cDNA
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Length-Limited (8 to 20) Oligomer Search GenBank Hit Table cDNA
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Length-Limited (8 to 20) Oligomer Search GenBank Hit Table cDNA
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Length-Limited (8 to 20) Oligomer Search GenBank Alignments cDNA
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Score/Length GenBank Hit Table Against cDNA: 8-20-mers down to 80%
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Score/Length GenBank Hit Table Against cDNA: 8-20-mers down to 80%
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Score/Length Alignments Against cDNA: 8-20-mers down to 80%
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Score/Length Alignments Against cDNA: 8-20-mers down to 80%
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QUESTIONS?
Michael P. Woodward
Supervisory Patent Examiner - Art Unit 1631
571 272 0722
[email protected]
John L. LeGuyader
Supervisory Patent Examiner - Art Unit 1635
571 272 0760
[email protected]
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