Mapping and PCR Exercise

This is a very short exercise on the use of MAP for restriction mapping and PRIME for PCR primer design.

First, FETCH SYNINCALAC (a plasmid cloning vector).

Use MAP to find all BamHI, EcoRI, HindIII, PstI, and KpnI restriction sites (use the -NOCOMP option to suppress the complement of the sequence )

Use MAP again, but this time use the -TABLE option to show the map as a list of cut positions).

Together, these two views provide a fairly good picture of the sequence. MAPPLOT provides a more graphical view - but of course you need to be able to view GCG graphics (either VersaTerm, or GCG Figure on a Mac, or use the FIGURE program to turn figures into printable Postscript - none of these options is available in the library computer room). MAPPLOT does have an option to create a plain text output file, but it is not any nicer than the output of MAP.

Try:

 >  mapplot -outfile=lac.plot  synincalac.gb_syn

(use the same set of enzymes as above)

Then view the output:

 > emacs  lac.plot

OK, now lets design some PCR primers.

First, look at the annotation for the Synincalac.Gb_Syn sequence.

Lets design primers to amplify the beta lactamase gene:

CDS             1854. .2714
                     /note="25 micrograms/ml ampicillin does not inhibit
                     growth, but 50 micrograms/ml causes some growth
                     inhibition"
                     /citation=[12]
                     /codon_start=1
                     /product="beta-lactamase"
                     /db_xref="PID:g208646"

The coding sequence runs from 1854 to 2714, so PCR primers should be located in the regions just flanking this sequence, lets say 1750-1850 for the forward primer and 2720-2820 for the reverse.

Use PRIME to find primers in these regions. From the GCG Manual section on PRIME, note that to specify a region to be included in the PCR product, use the command line option:

 -BEGin2=1854 -END2=2714   [target range for PCR amplification]

and specify the following parameters when prompted by PRIME:

                  Begin (* 1 *) ?  1750
                End (*  7922 *) ?  2820

 Minimum product length (* 100 *) ?  861
 Maximum product length (* 860 *) ?  1070

Okay, now something a bit more realistic.

Lets say that you need to amplify the first two exons of the BRCA2 gene in order to assay for mutations. Design two pairs of primers that can be used together in a 68 0C PCR reaction to amplify both fragments at once (a multiplex PCR).

Lookup BRCA2 in the UCSC Genome Browser and download the genomic sequence plus some upstream flanking seqquence.

The exons are in capital letters (make your primers just outside the exons) Send me your two pairs of primers: browns02@med.nyu.edu