Automated Searching of Polynucleotide Sequences

1
Automated Searching of Polynucleotide Sequences

• Michael P. Woodward
• Supervisory Patent Examiner - Art Unit 1631
• 571 272 0722
• michael.woodward_at_uspto.gov
• John L. LeGuyader
• Supervisory Patent Examiner - Art Unit 1635
• 571 272 0760
• john.leguyader_at_uspto.gov

2
Standard Databases

• GenEMBL .rge
• N_Genseq .rng
• Issued_Patents_NA .rni
• EST .rst
• Published_Applications_NA .rnpb

3
Databases at Time of Allowability

• Pending_Patents_NA_Main .rnpm
• Pending_Patents_NA_New .rnpn

4
Types of Nucleotide Sequence Searching

• Standard (cDNA)
• Oligomer
• Length Limited Oligomer
• Score over Length

5
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
  parameters-Gap Opening Penalty Gap Extension
  Penalty of 10
• 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.

6
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.

7
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

8
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

9
Standard Oligomer Searching

• the search parameters are the default
  parameters-Gap Opening Penalty Gap Extension
  Penalty of 60-mismatches not tolerated
• Consequently inefficient means of finding small
  sequences, and with lt100 in correspondence

10
Claim 1

• An isolated polynucleotide comprising SEQ. ID.
  No 1.

11
Searching Claim 1

• A standard search looking for full length hits is
  performed.

12
Standard (cDNA) search result
0001 CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA
CAGGAAACAGATGG 0060 2031 CGGCTCGTATGTTGTGTGGAATTGT
GAGCGGATAACAATTTCACACAGG---CAGATGG 2090
13
Claim 2

• An isolated polynucleotide comprising at least 15
  contiguous nucleotides of SEQ. ID. No 1.

14
Searching Claim 2

• An standard oligomer search is performed with an
  oligomer length of 15 nucleotides set as the
  lower limit for a hit.

15
Oligomer Search Results

• Standard Oligomer
• CAAATGCAGGCCCCCGGACCTCCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG
• Query CCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG 0060
• Database CCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG 2500
• Length Limited Oligomer
• CAAATGCAGGCCCCCGGACCTCCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG 
• Query CCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG 0060
• Database CCCTGCTCCTGGCTTTCGCCCTGCTCT
  GCCTGCCCTGG 0039
•  

16
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)

17
Searching Claim 3

• Seq ID No 2 is searched against the Polypeptide
  databases and it is back translated and
  searched against the polynucleotide databases.

18
Claim 4

• An isolated polynucleotide comprising a
  polynucleotide with at least 90 identity to SEQ
  ID No 1.

19
Searching Claim 4

• A standard search looking for full length hits is
  performed.
• Hits having at least 90 identity will appear in
  the results.

20
Claim 5

• An isolated polynucleotide comprising a
  polynucleotide which hybridizes under stringent
  conditions to SEQ ID No 1.

21
Searching Claim 5

• A standard oligomer search is performed as well
  as a standard search.

22
Searching Small Nucleotide Sequences

• John L. LeGuyader

23
Types of Small Nucleotide Sequences Claimed

• 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

24
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

25
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

26
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

27
Why doesnt 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 lt100 correspondence

28
Why doesnt 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

29
Standard Oligomer Searching

• Wont 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 lt100 in correspondence
• Better suited to finding long sequences

30
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 lt100 in correspondence
• Better suited to finding small sequences with
  100 correspondence

31
Score Over Length Searching

• Small oligos with lt100 correspondence
• within requested length and identity (gt60) range
• Manual manipulation of first 65,000 hits
• necessitates 2 additional hrs. of searchers
  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

32
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.

33
Mud Loach Growth Hormone cDNA

• 670 nucleotides long
• 630 nucleotides in the coding region
• 210 amino acids

34
Standard Search GenBank Hit Table Against cDNA
35
Standard Search GenBank Hit Table Against cDNA
36
Standard Search GenBank Alignments Against cDNA
37
Standard Search GenBank Alignments Against cDNA
38
Oligomer Search GenBank Hit Table Against cDNA
39
Oligomer Search GenBank Hit Table Against cDNA
40
Oligomer Search GenBank Alignments Against cDNA
41
Oligomer Search GenBank Alignments Against cDNA
42
Length-Limited (8 to 20) Oligomer Search GenBank
Hit Table cDNA
43
Length-Limited (8 to 20) Oligomer Search GenBank
Hit Table cDNA
44
Length-Limited (8 to 20) Oligomer Search GenBank
Alignments cDNA
45
Score/Length GenBank Hit Table Against cDNA
8-20-mers down to 80
46
Score/Length GenBank Hit Table Against cDNA
8-20-mers down to 80
47
Score/Length Alignments Against cDNA 8-20-mers
down to 80
48
Score/Length Alignments Against cDNA 8-20-mers
down to 80
49
QUESTIONS?

• Michael P. Woodward
• Supervisory Patent Examiner - Art Unit 1631
• 571 272 0722
• michael.woodward_at_uspto.gov
• John L. LeGuyader
• Supervisory Patent Examiner - Art Unit 1635
• 571 272 0760
• john.leguyader_at_uspto.gov