Step 6 - Sequencing analysis

a) Use a SeqEd layout showing vector sequences to look at forward sequence. Find the vector and linker at the front end. You can also use Sequencher and use motif to highlight the linker sequence. See how much insert sequence there is before the repeat. Sometimes there is none (ie the repeat starts right after the linker or there may even be no linker). If there is no front sequence, you're done and the locus is not going to be useful. Determine if there is a repeat and what size it is. If repeat is less than 12 repeats then you're done. If the sequence reads well through the repeat, find the linker and vector on the back side. See how much insert sequence there is after the repeat before the linker. It may be necessary to sequence the clone with the reverse primer as clones do not always sequence well through the repeat region.

b) Filemaker the Tilapia marker database. Make a new record. Enter the clone number, gel date, repeat size, front and back sequence lengths into page. Enter PCR band size on " more screening info page" to calculate the insert size. Sometimes it is helpful to characterize the repeat if it is complex in the comments box.

c) If the sequence is of high quality both before and after the repeat (and you can find the back vector or there is sufficient high quality sequence that there is >100 bp for designing a reverse primer) copy the sequence into the database.

d) Note whether reverse sequencing is necessary. If sequence is high quality and in database , note that this clone is "Done". If reverse sequencing is necessary, note reverse seq needed as "Yes" If there is a lack of front sequence or repeat is short or there is other problem, note this as "No".

e) Save the layout.

Step 7 - Primer design

Copy the sequence into the Primer3 program (Whitehead web page) and enter clone number. Bracket the repeat with [ ]. Leave the defaults as is (60C annealing temp etc). Let the program design primers. Check the chosen primers to see that they are not in highly repetative sequence. Copy the Primer 3 output to a Simple Text file. Open the Seq Ed file and locate the primers in the sequence. (Note : reverse primer is listed 5'-3' for opposite strand at top of primer3 output and won't be found that way in Seq ed file - copy sequence highlighted lower in output). Check that the sequence is high quality in the primer region.

Save Primer3 output file. Copy primers to database making sure to copy primers from top of output file.

The default PCR product size is 200 bp. If all the primers are designed to this length, it will be difficult to multiplex loci. Try to vary some of the lengths. Some of the products will naturally end up shorter or longer. It would be ideal to have 1/3 which are <150 bp, 1/3 which are 150-200 bp and 1/3 which are 200-250 bp. Products which are >300 bp will take a long time to run on genotyping gel.

Step 8 - Primer order and testing