Manual

Nucleotide Sequence Molecular Weight Calculator:

This program allows you to calculate the molecular weight of DNA and RNA molecules from sequence files which are written in the Genbank format (text format). The program can be used to calculate single- or double stranded sequences, or the molecular weight of the complement of a sequence. You can select the 3' and 5' terminus chemistry, and add common counterions in molar ratios to each residue.

Note: This program will utilize the database backend of UltraScan. This functionality allows you to save certain files and data in a database, among them the nucleotide files analyzed with this module. If the database system is not supported or available on your system, you will get a warning message when opening this module.

You can download DNA/RNA sequences directly into the calculator from Genbank. Clicking on the "Download Sequence" button will connect you to the Entrez database for downloading Genbank sequence files.

If you would like to import your own sequence, you need to make sure that the file is in the proper Genbank format.

Example sequence:

DEFINITION  exon from: Gallus gallus egg white lysozyme gene, partial cds.
ORIGIN
   1 agtcccgctg tgtgtacgac actggcaaca tgaggtcttt gctaatcttg gtgctttgct
  61 tcctgcccct ggctgctctg gggaaagtct ttggacgatg tgagctggca gcggctatga
 121 agcgtcacgg acttgataac tatcggggat acagcctggg aaact
  //

The following four fields are required:

Field 1: "DEFINITION"
This field has to be spelled in upper case and everything following it on the same line will be printed as the sequence title in the program.

Field 2: "ORIGIN"
This field has to be spelled in upper case and should be the only word in this line. This word indicates that the next line will contain the beginning of the actual sequence.

Field 3: The actual sequence (multiline). The sequence elements (a, g, c, t, u) can be spelled in upper or lower case, and can contain spaces. Each line with sequence data needs to contain exactly one string, separated by white space, in front of the actual sequence data. This is ordinarily the position of the first residue listed in that line.

Field 4: The sequence terminator "//". This terminator symbol should be on a separate line following the last sequence line. Any information past the terminator symbol is ignored.

Amy other information contained in the file is ignored.

The program uses the following algorithm (Ref.: Steve Hardies, University of Texas Health Science Center at San Antonio, Dept. of Biochemistry):

Natural isotope adjusted MWs

C	12.011
N	14.0067
O	15.9994
H	1.00794
P	30.9738

Adenine C5N5H5
Guanine C5N5OH5
Thymine C5N2O2H6
Cytosine C4N3OH5
Uracil  C4N2O2H4

ribose C5O5H10
deoxyribose C5O4H10

phosphate PO4

Adding the base to the ribose involves loss of 1 H2O 
Bonding the phosphate to either 5' or 3' OH causes loss of 1 HO

An internal ribonucleotide is then calculated as base+ribose+phosphate
-H2O - 2 OH.

MW of an internal ribonucleotide:

dA - 313.21
dC - 289.19
dU - 290.17
dG - 329.21
dT - 304.63

to make RNA, add 15.99 for each nucleotide
to make terminal 3' OH add 17.01
to make terminal 3' phosphate add 94.97
to make terminal 5' phosphate - no change
to make terminal 5' OH subtract 77.96

Does not account for the 2nd dissociable H of the terminal phosphate, which 
has a pK somewhere around neutrality. It would be 7.2 for a free phosphate.

Explanation for fields and buttons:

This group of functions allows you to select if the selected molecule is to be calculated as an RNA or DNA molecule, if the molecular weight of the sequence should be calculated including the molecular weight of the complement (in that case select "Double Stranded"), and what groups are on the 3' and 5' termini of the sequence.

If this option is selected, the complementary sequence will be generated, and the molecular weight of the complement only will be reported.

By clicking on the arrow buttons (single arrow: steps of 0.01, double arrow: steps of 0.1) you can select the molar ratio of counterions per nucleotide of the sequence. For example, if you selected a sodium ratio of 0.01, one in a 100 nucleotides of the sequence would be considered as having one sodium ion bound, and the weight of that ion would be added to the total molecular weight. If the sequence contained less than a hundred nucleotides, the corresponding fractional molecular weight of a sodium ion would be added to the reported molecular weight.

These fields show the first line of the sequence identification listed in the sequence file, as well as the molecular weight and sequence composition. If you are loading a DNA sequence, but selected the "RNA" option above, an warning message will be shown. An equivalent error message is shown if you selected "DNA" but loaded an RNA sequence.

Select Investigator: When searching for nucleotide sequences from the database, you can limit your search to only those sequences belonging to a specific investigator in the database. When you click on this button, the investigator selection dialog will open up and allow you to select the desired investigator. When the sequence is loaded from the database, this option is disabled, since an investigator is already associated with the selected sequence. Also, when committing a sequence to the database an investigator needs to be chosen to be associated with the sequence.

Sequence Loading:
You can load sequences into this program by one of two methods:

Load Sequence from HD
Use this button to load an ASCII sequence file from your harddrive. A file dialog will open and allow you to search for *.seq files.

Query Sequence from DB
If you are using a database backend for UltraScan you can query the database for nucleotide sequence files that have previously been committed to the database. Sequence files available in the database will be listed in the listbox once you click on this button. More information about various database functions is available elsewhere.

Download Sequence: Download a sequence from the Genbank database.

Save Sequence to DB: Use this function to save a sequence to the database. Before storing a sequence in the database, you need to have a copy of the sequence stored locally, for example, on your harddrive. You can then select the sequence to be stored in the database from a filedialog and after selection, commit the sequence to the database.
Calculate: Update the molecular weight calculation and apply any ion changes, if selected.
Save Result: Save the result of the calculation to a file. This file will be saved in your results directory. The file will be named by the name the sequence was stored, and a suffix of ".nucleotide_res" will be added.
Delete Sequence from DB: Delete the selected sequence from the database. This function requires administrator privileges.
Reset: Clear the loaded sequence from memory
Help: Show this help file
Close: Close this function

www contact: Borries Demeler

The latest version of this document can always be found at:

http://www.ultrascan.uthscsa.edu

Last modified on January 12, 2003.

	This group of functions allows you to select if the selected molecule is to be calculated as an RNA or DNA molecule, if the molecular weight of the sequence should be calculated including the molecular weight of the complement (in that case select "Double Stranded"), and what groups are on the 3' and 5' termini of the sequence.
	If this option is selected, the complementary sequence will be generated, and the molecular weight of the complement only will be reported.
	By clicking on the arrow buttons (single arrow: steps of 0.01, double arrow: steps of 0.1) you can select the molar ratio of counterions per nucleotide of the sequence. For example, if you selected a sodium ratio of 0.01, one in a 100 nucleotides of the sequence would be considered as having one sodium ion bound, and the weight of that ion would be added to the total molecular weight. If the sequence contained less than a hundred nucleotides, the corresponding fractional molecular weight of a sodium ion would be added to the reported molecular weight.
	These fields show the first line of the sequence identification listed in the sequence file, as well as the molecular weight and sequence composition. If you are loading a DNA sequence, but selected the "RNA" option above, an warning message will be shown. An equivalent error message is shown if you selected "DNA" but loaded an RNA sequence.
	Select Investigator: When searching for nucleotide sequences from the database, you can limit your search to only those sequences belonging to a specific investigator in the database. When you click on this button, the investigator selection dialog will open up and allow you to select the desired investigator. When the sequence is loaded from the database, this option is disabled, since an investigator is already associated with the selected sequence. Also, when committing a sequence to the database an investigator needs to be chosen to be associated with the sequence.
	Sequence Loading: You can load sequences into this program by one of two methods: Load Sequence from HD Use this button to load an ASCII sequence file from your harddrive. A file dialog will open and allow you to search for .seq files. Query Sequence from DB* If you are using a database backend for *UltraScan* you can query the database for nucleotide sequence files that have previously been committed to the database. Sequence files available in the database will be listed in the listbox once you click on this button. More information about various database functions is available elsewhere.
	Download Sequence: Download a sequence from the Genbank database. Save Sequence to DB: Use this function to save a sequence to the database. Before storing a sequence in the database, you need to have a copy of the sequence stored locally, for example, on your harddrive. You can then select the sequence to be stored in the database from a filedialog and after selection, commit the sequence to the database. Calculate: Update the molecular weight calculation and apply any ion changes, if selected. Save Result: Save the result of the calculation to a file. This file will be saved in your results directory. The file will be named by the name the sequence was stored, and a suffix of ".nucleotide_res" will be added. Delete Sequence from DB: Delete the selected sequence from the database. This function requires administrator privileges. Reset: Clear the loaded sequence from memory Help: Show this help file Close: Close this function