New PCR primer analysis program

J. Michael Cherry cherry at frodo.mgh.harvard.edu
Fri May 8 10:47:00 EST 1992


A new Macintosh application for use in PCR reactions was announce on the
info-mac list. Below I've included a text version of this programs 
documentation. You can obtain a copy of Amplify from 
sumex-aim.stanford.edu via anonymous ftp. Look for the file:

      /info-mac/app/amplify-10.hqx

I'm sure the various molecular biology archive will be picking up the
Compactor archive of Amplify very soon. If you can't get to sumex I've
also put the amplify-10.hqx file on amber.mgh.harvard.edu in the mac
directory, ("cd mac" then "get amplify-10.hqx").

The author, Bill Engels, can be reached at WREngels at wisc.macc.edu

--------------------------------------------------------------------------
Amplify  (C) 1992  Bill Engels

I. Introduction

This software is for use in designing, analyzing, and simulating
experiments involving the polymerase chain reaction (PCR).  PCR is a
technique used by molecular biologists to amplify highly selected
segments of DNA.  If you never heard of PCR, you probably have no use
for this program.  If you want to know more about the process, you can
look up the book, PCR Technology, by H.A.  Erlich (1989, Stockton
Press). 

Very briefly, PCR will take a short stretch of DNA (usually fewer than
3000 bp) and increase its copy number about a million fold so that one
can determine its size, DNA sequence, etc.  The particular stretch of
DNA to be amplified, called the target sequence, is identified by a pair
of DNA primers, which are even shorter pieces of DNA (usually about 20
bp) that have been synthesized in large quantities.  To use Amplify you
must supply the sequences of the primers and the target DNA.  The
software then analyzes the combination of primers and target sequence
you have chosen to determine what portions of the target are likely to
be amplified.  It also provides various bits of helpful information
about the reaction products, primer binding sites, etc., that can help
in planning a PCR experiment and designing primers. 

II. Quick Tutorial

1.  Boot up Amplify by double-clicking on the file Primers.pri.  You
should now be faced with a window showing the Amplify logo.  Close it to
reveal another window with a list of primers.  (This is just list of
primers that is currently available in my lab.) The primer sequences are
in the first column and their names are in the second. 

2.  Find the primer named 1700 and place the cursor anywhere in that
line.  Then select Use this Primer from the menu, or hit -1.  That will
open another window, and add primer 1700 to the list of primers that
will be used.  Then do the same for primer 2230. 

3.  Next, open a file of target DNA by selecting Open Target Sequence
from the File menu, or by hitting -O.  Find the file called
P Element.seq.  (This is the sequence of the P transposable element in
Drosophila melanogaster.  )

4.  You have now specified everything needed for a PCR experiment.  So
pick Run PCR ( -3) from the PCR menu.  A map showing the PCR products
will then be generated as follows:

5.  The arrowheads indicate points of primer binding (darker means a
better match), and the bars below the sequence are potential amplified
fragments (heavier bars are expected to amplify better).  Explore this
result by clicking on the various elements of the map.  In each case,
you will get an information screen with data about the item you clicked. 
Note that the lighter arrowhead for primer 1700 indicates a weak match
that is probably not desirable in the experiment.  Click on it to see
how well the primer matches the target sequence. 

6.  To get a closer look at just a portion of the sequence, you may zoom
in on any chosen section of the map.  To do this, hold the command ( )
key down and drag with the mouse to draw a rectangle around the region
of interest.  Then select Zoom In from the PCR menu.  You can repeat the
process any number of times to get even higher resolution, or else
select Zoom Out to get back to the original scale. 

III. Working With Primer Lists

To use Amplify you should keep a text file listing all the primers
available to you.  You can create such a list with Amplify, or else use
any word processing or spreadsheet program.  Each line of the list
should start with the sequence of the primer (Always in the 5+ to 3+
direction) followed by a tab and then the name of the primer.  The name
should be short so that it will fit on the map.  You can also append
additional information about the primer on the same line after another
tab.  Amplify can accept any text file as the primer list.  However, if
you wish to have a double-clickable file, you must create it with
Amplify's Save AsI command.  In addition, the name of the file should
end with .pri so that Amplify will know that it is not a target
sequence file. 

You can edit your primer list in Amplify's Primer List window just as
you would edit in any Macintosh text window.  In addition, you can use
the Edit menu to interchange between upper and lower case letters in
order to identify any particular part of the sequence.  You can also
reverse the polarity of any sequence: select the portion you want to
reverse, then pick Reverse Sequence from the Sequence menu.  This action
takes the complementary strand and displays it in the 5'->3' direction. 
Only the primers listed in the Primers In Use window will be involved in
the amplification.  You can move primers from the main list to the In
Use list by copying and pasting or by picking Use This Primer ( -1) with
the cursor on the primer you want to select.  To remove a primer from
the In Use list, you can use the Remove This Primer menu item in the
same way, or else simply erase it as you would any text. 

IV.  Working With The Target Sequence

  Amplify can read DNA sequence information from any text file.  If you
create a text file with Amplify's Save AsI command, it will have the
Amplify icon and be double-clickable to open the Amplify program. 

The target sequence text can have an optional header section before the
actual sequence starts.  This header can consist of anything you like
provided it ends with two periods (..).  Anything following the ..
is taken to be part of the sequence.  If you wish to use a header that
includes .. as part of the text, you can use the Preferences command
to change the header delimiter to any two-character string.  Whenever
you use the target sequence, Amplify attempts to put the DNA into a
standard format.  It removes any characters other than A, T, C and G,
plus the corresponding lower-case letters and arranges the sequence in
80-character lines.  This reformatting is done only to the sequence part
of the text, and not to the header.  You can change the number of
characters per line to something other than 80 with the Preferences
command. 

You can edit the target sequence window as you would any Macintosh text. 
You can also use the upper- and lower-case commands as well as the
Reverse Sequence command as described previously in part III. 

You can search the sequence using the Find Pattern ( -F) command.  This
command is better for searching sequences than the usual find command
available in most text editors, including the one in the Edit menu of
Amplify.  With the Find pattern command you can search in both
orientations if you wish, and line breaks are ignored in the search.  In
addition, you can specify a maximum number of allowable mismatches in
the search pattern.  Once you have found the pattern, you can find the
next occurrence with the Find again ( -G) command.  Use the Select
SequenceI command to select a stretch of sequence according to its
coordinates, and the Get sequence info ( -I) command to see size, base
composition, etc., of any selected portion of your sequence. 

V.  The Run PCR command

The Run PCR ( -3) command is what does the real work.  Before you use
this command, you must have loaded a target sequence and put at least
one primer into the Primers In Use window.  The Run PCR command performs
the following series of actions:

1.  The target sequence is formatted as described above.  If it has
already been formatted and no editing has been done since then, this
step is skipped. 

2.  Each selected primer is checked against itself and all the others to
determine if any are likely to form dimers. 

3.  The target sequence is searched in both directions for matches to
the primers you have selected.  Two criteria called primability and
stability are used to estimate how likely a given match is to
contribute to the amplification reaction.  More about these later. 

4.  Each primer match that qualifies under both criteria is used to
screen for possible amplified fragments.  Each such fragment is
evaluated to determine its probable degree of amplification depending on
the quality of the matches involved, the length of the sequence, it's GC
content, etc. 

5.  The results of this analysis are displayed graphically to show the
matches and the amplified fragments.  If any potential dimer-forming
pairs are identified, they are shown also. 

VI.  Working with the amplification map

Matches and Fragments

The target sequence is represented as a horizontal line with tick marks
every 100 bp.  Any matches between the primers and the target sequence
are shown as arrowheads.  Rightward-directed matches are above the line
and leftward ones below.  Segments of the target sequence that are
candidates for amplification are shown as bars below the target
sequence.  They are drawn starting with the RbestS fragments on top,
i.e., those expected to have the greatest abundance following the PCR. 
The primer matches and amplified fragments are drawn so that darker
fills and heavier lines correspond to better matches or amplification. 
This scheme is



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