• Antibodies
• Purified proteins
• qPCR Primers

• SeqIt is our web-based program for rapid database searching using amino acid sequences.
• NucIt is a web-based system that performs oligonucleotide primer design, degenerate oligonucleotide primer design, heteroduplex analysis, repeat searches, DNA translation and siRNA duplex design.
• µArrayDB is an internet-based DNA microarray database with project management and data archive features designed especially for DNA microarray core facilities.
• ArrayQuest is our system for web-based analysis of DNA microarray data.
• GeneMESH is a web-based system for identifying intermolecular interactions that exist among a set of proteins finding genes associated with a given biological term and grouping genes according to a MeSH category.
• OligoInventory is a web­-based LIMS program for archiving oligonucleotide records.
• Frozen Cell Inventory is a web-based LIMS program for managing frozen storage of cell lines.
• WebDCAS is a configurable web-based system for managing scientific information.
• Protein Molecular Weight Calculator. Free
• Extinction Coefficient Calculator. Free
• Molar Concentration Calculator. Free

SeqIt™ Search Help

• The on-line version of SeqIt™ searches a database having approximately 200,000 protein sequences.
• SeqIt will format sequence alignments to colorize specific residues
• SeqIt™ query sequence entry can include a range of permutations such as unknown residues in a particular position (e.g., X or ? = any residue) or a variety of residues occurring in a particular position (e.g., K or R = (KR)) or a variable number of residues occurring between particular residues (e.g., 4-5X = 4x or 5X; ... = 3X). In addition, specified residues can be excluded in a particular position (e.g., {KR} = any residue except K and R). The query sequence entry format is case insensitive. The search can also identify proteins having sequences that match exactly or with varying degrees of mismatch.

  Syntax quick reference:

  • X or ? = any residue
  • (ST) = S or T
  • 3A = AAA
  • 4-5X = 4X or 5X
  • {KR} = not (KR)
  • ... = 0-3X
  • entry format is not case sensitive.
• SeqIt™ search results are formatted as follows: Protein sequences containing the query sequence are displayed on the right si de of the table. The query sequence motif will be flanked by a number of residues specified by the user. Multiple sequences containing the query sequence are aligned with each other. Unique identifiers for each protein sequence (e.g., accession number (AC)) are presented and hypertext-linked to  ExPASy to allow easy retrieval of pertinent information. The number of amino acid residues (AA) contained within each protein are indicated as is the location of the query sequence within each protein (POS).
• Subset searching: Protein sequences identified through a given search are stored as a subset file and can be searched separately by selecting Subset under database source selection. The subset grows with each dataBase search performed. The subset can be reset or cleared, prior to a dataBase search.

• Speed of on-line SeqIt™ searching: A bench mark search for a 5 residue sequence (RKH) G (DE) (ST) P currently takes about 5 seconds to complete. However, if another search is in progress you may experience delays. The server accepts requests on a first-in-first-out (FIFO) basis. After about one minute the server may tell you to check back in to retrieve your query results.

  To check back in and obtain your query results stored on the server, go to the SeqIt™ search page and type your E-mail address into the third form area, and click the submit button. The last search that you performed will be accessible by then selecting "LASTFIND.HTM"

• Colorized amino acid sequence alignment: This feature enables users to specify which residues will be colorized in the output alignment. Chemically conserved amino acids (e.g., R, K and H; S and T; D and E etc) have the same color. To use this feature choose the file "LASTFNDC.HTM". This feature works best with Microsoft Internet Explorer. Search results of greater than 50 hits may cause NetScape to error.

• Use your browser's Reloadfeature to assure current output views. This is important because your browser may display a cached page which may not be the current page on the server.

SeqIt™ Query Syntax Quick Reference

• Query Syntax

  X or ?	any residue
  (ST)	S or T
  3A	AAA
  4-5X	4X or 5X
  {KR}	not (KR)
  ...	0-3X

  Note: entry format is not case sensitive.
• Tip: try using dots to allow permutations, example: 'asd..cc' will search for 'asdcc' and 'asd xcc' and 'asdxxcc'

SeqIt™ SearchRegion Help

SeqIt™ Features

• Motif Search can be used to find user specified query sequences within an individual target protein sequence, all sequences in a database or in a subset of sequences. The query sequence can include a range of permutations such as unknown residues in a particula r position (e.g., X or ? = any residue) or a variety of residues occurring in a particular position (e.g., K or R = (KR)) or a variable number of residues occurring between particular residues (e.g., 4-5X = 4x or 5X; ... = 3X). In addition, specified residues can be excluded in a particular position (e.g., {KR} = any residue except K and R) The query sequence entry format is case insensitive. The search can also identify proteins having sequences that match exactly or with varying degrees of mismatch.

  After a search is completed protein sequences containing the query sequence are displayed on the left side of the output table. The query sequence motif will be flanked by a number of residues specified by the user. When multiple sequences are found to contain the query sequence they are aligned with each another, Unique identifiers for each protein sequence (e.g., accession number (AC)) are presented and accession numbers hypertext-linked to EXPASY to enable retrieval of pertinent information. The number of amino acid residues (AA) contained within each protein are indicated as is the location of the query sequence within each protein (POS). Align Motif can be used to manually optimally align sequences in a SeqIt™-generated alignment.

  Protein sequences identified through a given search can be stored as a subset file and can be searched separately using the Use Subset feature.

• Repeat Search can be used to find internal repeats in a target protein sequence.

• Load Sequence will load any amino acid sequence from a database using an accession number. Protein sequences can also be retrieved from a database using a name search. A sequence may also be pasted directly into the load sequence window.

• Diagonal Plot of Sequence Similarity can be used to display results from Repeat Search in a two-dimensional matrix plot.

• Bar Chart of Motif Frequency can be used after a Motif Search to display the frequency distribution of the query sequence in the target sequence.

• Biophysical Properties determines the amino acid composition, molecular weight and extinction coefficient (A280 0.1%) of a specified target sequence.

  SeqIt™ will create a Work Journal File of all activity that occurs during a given SeqIt™ session. The current and past Work Journal Files can be viewed during a session using the View command.

Primer Design locates optimal oligonucleotide sequences within a user specified target DNA by first searching both strands for occurrences of unique oligonucleotides of specified length. Unique sequences are then ranked on the basis of nucleotide matches with similar sequences in both strands of the target and by the relative stability (delta_G) of their exactly matched duplex hybrid. Ranked sequences are listed along with their position in the target, their %GC content , molecular weights, duplex melting temperature (Tm) values and free energy of self complementation values. NucIt™ determines and reports Tm values based on three different equations (see Tm calculator section for equations). Output Tm values are arranged into columns adjacent to each respective sequence (with the far right Tm derived from equation 3, the Tm to its left derived from equation 2, and the far left Tm derived from equation 1). If you are going to use the Tm values based on thermodynamic near est-neighbor parameters (equation 3) then the molarity of monovalent cation [Na+] and the oligomer probe concentration ([CT]) need to be calculated and entered. Tm values based on equation 3 are considered more accurate than those based on 1 and 2 when the two variables ([Na+] and [CT]) are computed accurately.

The first oligonucleotide sequences in an output list have the lowest degree of matching between them and similar sequences found elsewhere in the target sequence. These oligonucleotides will also have lower delta_G values and thus higher relative duplex stability when paired with their exact complementary target. An oligonucleotide that has 10 out of 20 matches with another site is considered a better potential primer than one having 11, 12, 13 ... matches with similar sequences in the target. Conversely, an oligonucleotide that has 10 of 20 mismatches with another site is considered better than one with 9, 8, 7.out of 20 mismatches. Oligonucleotides at the beginning of the list are considered to be the best choices as compared with those at the bottom of the list. Primers further down the list have higher content of nucleotides that match elsewhere in the target. Don't think that primers further down a list are not suitable choices. For each potential primer consider its duplex melting temperature (Tm) with a similar sequence elsewhere in the target. You can assume that each mismatch will cause a 1-1.5°C difference in the Tm. To examine the other occurrences of sequences within a target that have a high degree of similarity with a particular oligonucleotide, use the OTHER OCCURRENCES feature.

For polymerase amplification experiments requiring two oligonucleotide primers from opposite strands of a target, use the list to select two oligonucleotides of the desired distance from one another and most importantly whose Tm 's are similar. Remember to take the reverse complement of the downstream primer. When two oligonucleotides are selected from the listing they should be then entered into the COMPARE OLIGOS function to determine whether the two have duplex complementarity (extensive complementarity at the 3' ends is not desired). When a given oligonucleotide sequence is selected from the PRIMER DESIGN listing it should also be entered into the Tm CALCULATOR function to check its degree of self-complementarity and its ability to form a hairpin structure. Note: For DNA amplification we recommend that the annealing segment temperature be set to 3-5°C below the predicted Tm of the primer with the lowest Tm of the selected pair.

An important feature of PRIMER DESIGN is the ability to define a region of the target DNA to perform the analysis on. Limiting the size of the window often shortens the time required to analyze the target.

Using the INVERSE command, analyses can be made on the reverse complement of any sequence. The orientation of any loaded sequence will appear next to the sequence name as either "Forward or Reverse/Complement".

Tm Calculator estimates the melting temperature (Tm ) of a hybrid formed between a specified oligonucleotide probe and its target based on the following equations:

1. Tm = 2°C (A + T) + 4°C (G + C) (Itakura et al., 1984 and Suggs et al., 1981), for oligonucleotides shorter than 18 bases.

2. Tm = 81.5°C + 16.6(log10 [Na+] ) + 0.41(%G+C) - 0.63 (%formamide) - (300 + 2000 ([Na+]))/l (Bolton and McCarthy, 1962). Where (%G + C) = percentage of guanine plus cytosine nucleotides; [Na+] = the molarity of monovalent cation; and l = the length of the hybrid duplex in bases. The equation is valid over a [Na+] range of 0.01 - 0.4 M for oligonucleotides 14-70 bases in length. The program prompts the user to enter an estimated molar [Na+] value for the annealing and the percentage of formamide (if applicable).

3. Tm = F1 - 273.15 + 16.6(log10 [Na+]) - 0.63 (%formamide). (Freier et al., 1986 and Breslauer et al., 1986 using nearest-neighbor thermodynamic parameters). Where F1 = delta_Ho / [ delta_So + R x ln(CT) ]; R =1.987 cal / (oK x mol). CT is the total (molar) concentration of oligomer probe (for non-self complementary oligonucleotides CT in the equation is replaced by CT /4). [Na+] = the molarity of monovalent cation.

For DNA oligonucleotides, 14 - 18 bases in length, Tm CALCULATOR displays values based on all three equations (adjacent to output Tm values are the references to the equations used for computing the Tm). For DNA oligonucleotides greater than 18 bases in length, the program displays estimated Tm values based on equations 2 and 3 as the use of equation 1 results in an overestimation of the Tm of hybrids longer than 18. Calculations based on equations 2 and 3 require input of [Na+]. Calculations based on equation 3 require input of [CT]. Default settings are 0.07 M for [Na+] and 1.0 uM for [CT]. For duplex melting temperature calculations involving RNA/DNA hybrids, only equation 3 is used.

The thermodynamic parameters for hybrid duplex formation, enthalpy and entropy changes (delta_Ho and delta_So), are also tabulated and reported. In addition, the temperature of dissociation (Td) for filter hybridization is calculated with the temperature correction (-7.6°C) used as described by Rychlik and Rhoads (1989). For each oligonucleotide analyzed, its length, percent GC content and molecular weight values are displayed.

References
Bolton J. L. and B. J. McCarthy (1962) A general method for the isolation RNA complementary to DNA. Proc. Natl. Acad. Sci. 48:1390.

Breslauer K. J., R. Frank, H. Blocker and L. A. Marky (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. 83:3746-3750.

Freier S. M., R. Kierzek, J. Jaeger, N. Sugimoto, M. H. Caruthers, T. Neilson and D.H. Turner (1986) Improved free-energy parameters for predicitions of RNA duplex stability. Proc. Natl. Acad. Sci. 83:9373-9377.

Itakura K., J. J. Rossi and R. B. Wallace (1984) Synthesis and use of synthetic oligonucleotides. Ann. Rev. Biochem. 53:323.

Rychlik W. and Rhoads R. E. (1989) A computer program for chosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA, Nucl. Acids Res. 17:8543-8551.  [Abstract]

Suggs S. M., Hirose T., Miyake E. H., Kawashima M. J., Johnson K. I. and Wallace R. B. (1981) In D.D. Brown ed., ICN-UCLA Sympo. Develop. Biol., Using purified genes, Academic Press Inc. New York 23:683-693.

X-Variables allows the user to change or set default variables used in the equations for determining Tm , delta_Go, delta_Ho and delta_So values. Values may be entered in exponential form (e.g. to enter 70 mM type 70e-3, to enter 100uM type 100e-6). Note: The Temp variable used for computing delta_G does not affect Tm computations. The default oligonucleotide length is set at 18 bases.

Motif Search is designed to search target DNA sequences for specified sequences. The specified "motif" sequence may have a defined number of mismatches relative to the target. Motifs may contain either/or positions, wild positions and even positions that exclude particular residues. For example, if you are interested in searching for a sequence containing either an A or a T in a particular position you would enter the two bases in parenthesis (e.g. AAT(AT)AA). If you want to search for a motif in which specific residues were separated by a fixed or variable number of wild residues you merely enter X for each position or a range of variable residues (e.g. GCXATCA3-6XGAATTC).

Using the INVERSE command, motif searches can be made on the reverse complement of any sequence. The orientation of any loaded sequence will appear next to the sequence name as either Forward or Reverse/Complement.

Repeat Search is designed to search a target DNA sequence for direct repeats of specified length. A major feature of this program is that the user may define the number of allowable mismatches for a given sequence length. Using the INVERSE command, repeat searches can be made on the reverse- complement of any sequence.

DNA Translator translates a DNA sequence into a protein sequence using the Universal Genetic Code. The user can specify the nucleotide position to start and stop translation. The translation is output to the screen in the single letter amino acid code. Stops are indicated by dashes (-). For each translation the length of the longest open reading frame is displayed along with its start position. Using the INVERSE command, translations can be made on the reverse complement of any sequence.

Degenerate Oligonucleotide Primer Designer will determine the degenerate oligonucleotide sequence and the extent of its degeneracy for a given amino acid sequence. The program translates an amino acid sequence to DNA. This program aides in the design of oligonucleotide probes when only amino acid sequence is available for a given protein. Using the Universal Genetic Code and a specified amino acid sequence (up to 60 residues in length), NucIt™ derives the nucleic acid sequence with degenerate residue combinations indicated by I.U.B (International Union of Biochemistry) nucleotide codes (see below, the codes are also displayed as a legend in the output). The degeneracy value for each codon in the sequence is indicated. Based on the back-translated sequence NucIt™ will display a list of oligonucleotides of user specified length, ranked according to their total degeneracy.

NucIt™ uses I.U.B nucleotide codes for bases and degenerate combinations thereof: A=A, C=C, G=G, T=T, N=ACGT, V=ACG, M=AC, H=ACT, D=AGT, R=AG, W=AT, Y=CT, S=CG, K=GT, B=CGT

Other Occurrence finds all unique oligonucleotides of a specified length in a target sequence and then it scans the target sequence for the occurrences of similar sequences.

Licenses and Trademarks
The Polymerase Chain Reaction (PCR) process is covered by patents issued to Roche.

Also see "legal notices" for Terms of Use and Disclaimer for NucIt™.
Also see "legal notices" for Licenses and Trademarks.

Copyright Notice and Disclaimer
Copyright NucIt™ 1990, All Rights Reserved by Array Genetics.

Terms of Use and Disclaimer for NucIt™ This software is provided by Array Genetics, Inc. 'as is' and any express or implied warranties, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose are disclaimed. In no event shall Array Genetics, Inc. be liable for any direct, indirect, incidental, special, exemplary, or consequential damages (including, but not limited to, procurement of substitute goods or services; loss of use, data, or profits; or business interruption) however caused and on any theory of liability, whether in contract, strict liability, or tort (including negligence or otherwise) arising in any way out of the use of this software, even if advised of the possibility of such damage.

If you have any questions or comments about NucIt™ please  Email them to us.

 Elbashir et al. (2001)

License:

1. Use of NucIt Batch siRNA Duplex Design requires a license.
2. Please review our Terms of Use and Disclaimer for NucIt™
3. Please  inquire for pricing.

Features:

1. siRNAs identified by this algorithm are based on parameters described by  Elbashir et al. (2001).
2. Sense siRNA sequences are hyperlinked to enable BLAST searching.
3. the algorithm will flag potentially problematic siRNA sequences having polyG, polyC, polyA, and polyT nucleotides.

siRNA Design considerations:

1. It is recommended that you refer to the article by  Reynolds et al. (2004) for information pertaining to rational design of siRNAs.
2. siRNA duplexes should be composed of 21 nucleotides sense and 21 nucleotides antisense strands, paired so that each has a 2 nucleotides 3' dTdT overhang.
3. siRNA sequences should have low to moderate GC content. siRNA duplex designs are restricted to those having 30-70% GC content.
4. Sequences with more than three Gs or three Cs in a row should be avoided, because polyG and polyC sequences may hyperstack and thus interfere in the siRNA silencing mechanism. Design Flag legend: 3C-SS = CCC present in sense strand; 3G-SS = GGG present in sense strand; 3C-AS = CCC present in anitsense strand; 3G-AS = GGG present in antisense strand.
5. Delta_G calculations for hair-pin self complement and duplex formations use sense strand siRNA and antisense siRNA sequences in which 'U' nucleotides are substituted with 'T' nucleotides and assume that the thermodynamic properties are similar. Other strand folding formations may occur and potentially interfere in the siRNA silencing mechanism. However, these formations are not highlighted by this algorithm. Delta_G values of the sense and antisense strands are hyperlinked to their computational source  (NucIt melting temperature calculator).
6. To minimize the chance of selecting siRNAs with homology to non-targeted genes, the user is advised to search candidate siRNAs against the UniGene database using BLAST  (http://www.ncbi.nlm.nih.gov/BLAST/). Sense siRNA sequences are hyperlinked to enable BLAST searching. Remember to select the organism before starting the BLAST search.

Input Notes:

1. numbers and spaces within the input target sequence are ignored.