From: msalminen@nphi.fi
Newsgroups: bionet.molbio.hiv
Subject: Some HIV PCR primers
Date: 17 Aug 92 15:36:37 GMT
Organization: National Public Health Institute, Finland

Hello again!

Some time ago I suggested the start of a project that would compile PCR primers
useful in HIV-reserarch. At that time I had in mind a rather sophisticated
database that would present various data about the primers, such as the
references where they had been used, the optimal conditions they had been used
at, their sensitivity and specificity for different strains, the combinations
they could be used in etc, etc.

However as things ususally turned out, there was some interest, but I
underestimated the work to be done for the collecting of these data.

The result is that I decided to put my own primers (those I have used, not
necessarily designed) into a simple format which can be used by a useful
analysis program (I used Seqaid II format because I use it myself) and post
them to the net for anyone interested to use.

I have not given up my original notion about collecting primers, but will
continue it and work on with finding a suitable database format for them.
Meanwhile, I will send updates to this list whenever I have had the time to
compile more primers. The list is fairly short at the moment, but you will 
find useful primers for most of the genes in it.

To use the list, get a copy of Seqaid II by Roufa et Rhodes (MS-DOS), put the
file in the same directory as the program files (or in the path) as
hivprime.txt and give this filename as the source in the "search for sites"
section of the program. Note that not all of the primers recognize all
published HIV-isolates. You can get Seqaid II from the molbio archives by FTP,
for example nic.funet.fi should have a copy in the molbio directories.

The primers are presented in a self-explanatory format (+strand) and new can
easily be added using any ascii text editor. They can propably also be imported
to various other analysis programs with some editing.

When using the primers, you must naturally optimize each set individually, but
the following conditions may be a good start:

		95 C, 30 s
		55 C, 30 s
		72 C, 2 min

		30 cycles

	end	72 C, 5 min

		1 uM primer
		1.5 mM Mg2+
		200 uM Each dNTP
		Standard PCR buffer

You might also note that nested PCR and Hot-Start are a significant help,
however, BEWARE OF THE TERRIBLE CONTAMINATION PARASITE, so keep your stocks in
HIV-DNA free laboratories (its no joke!)

There is no guarantee that the primers will work, many of them however do for
me, but I wont take any responsibility for the use of them in any setting. I
strongly discourage anyone to use them for diagnostic purposes, their
sensitivity is not tested and the contamination problem easily leads to false
positives. Also I take no responsibility for typographic errors that I may have
introduced.

Hope that you will find the sequences useful in your work,

	Cheers, Mika

-------------------------- 8< cut here 8< ------------------------------------
:                   HIV PRIMER TABLE
:
:
:  Bases:   A,C,G,T, N=any base, Y=pyrimidine,  R=purine, M=A or C,
:           K=G or T, S=G or C, W=A or T, H=A, C or T, B=G, T or C,
:           V=G, C or A, D=G, A or T.
:
:
:  Comment Lines:  must begin with non-alphabetic characters in column 1.
:
:  Data Lines:  must begin with an integer (i.e. the number of bases in the
:               recognition site).
:
:
:      Insert new primers, grouped according to the number of bases
:  in the recognition site.  Use the line format:  number of bases in
:  the site, the site name, recognition sequence, offset from the
:  first base in the sequence to the base 5' of the 
:  primer end, optional 2nd sequence and offset.  No line in the
:  textfile can be longer than 80 characters, and no primer
:  site can be longer than 50 characters.  If a site is not
:  associated with an offset, the offset must be set to 0.  The
:  entire textfile can contain no more than 255 lines of data,
:  excluding comment lines (see above).  The table may be sorted but
:  must ALWAYS MAINTAIN AN ORDER ACCORDING TO THE NUMBER OF BASES IN
:  THE RECOGNITION SITE written in column 1.  Following a
:  primer site search, SEQAIDII reports sites as the base 5' of
:  the phosphodiester linkage cleaved.
:
:  These PCR primers do not necessarily recognize every HIV-1 isolate
:  they must be tested individually.
:
:                                                   Edited 28/4/92
:
15,ENV2        ,AGATAGCAAATTAAG,+15
:
16,BJGAG 5     ,CAGAGCCAACAGCCCC,+1
:
17,SK01GAG     ,CAGGGAGCTAGAACGAT,+1
17,SK02GAG     ,TCAGACAGGATCAGAAG,+17
17,SK30LTR     ,TAGAGATCCCTCAGACC,+17
:
18,GAG4        ,AGGAAGCTGCAGAATGGG,+1
18,SK32POL     ,ACCTGCCACCTGTAGTAG,+1
18,SK29LTR     ,ACTAGGGAACCCACTGCT,+1
18,HIV2SK89LTR ,AGGAGCTGGTGGGGAACG,+1
18,HIV1SK1OOGAG,ATCAAGCAGCCATGCAAT,+1
:
19,ENV1        ,GTAGTATTGGTAAATGTGA,+1
:
20,MSENV 1     ,GTATCCTTTGAGCCAATTCC,+1
20,MSENV 3     ,ATTGTGGAGGGGAATTTTTC,+20
20,MSGAG 7     ,GATGACAGCATGTCAGGGAG,+1
20,P1GAG       ,GAAGGAGAGAGATGGGTGCG,+1
20,P2GAG       ,GGGACAGCTACAACCATCCC,+20
20,GK56GAG     ,TTATCAGAAGGAGCCACCCC,+1
20,HIV2SK100GAG,ATCAAGCAGCCATGCAAGAT,+1
20,SK68ENV     ,AGCAGCAGGAAGCACTATGG,+1,
20,K4ENV       ,TTATTGTCTGGTATAGTGCA,+1
20,K5ENV       ,CTGGCTGTGGAAAGATACCT,+20
20,HTLVANEF    ,ATGCTGATTGTGCCTGGCTA,+1
20,HTLVBNEF    ,GGACTGGAAGGGCTAATTCA,+20
20,CO12TAT     ,CTTAGGCATCTCCTATGGCA,+1
20,CO11TAT     ,GAGGGAACCCGACAGGCCCG,+20
20,HIV2SK90LTR ,TGCTAGACTCTCACCAGCAC,+20
20,GK55GAG     ,AGGGGAAGTGACATAGCAGG,+20
:
21,BJGAG 3     ,GACAGGCTAATTTTTTAGGGA,+21
21,MSGAG 4     ,GACAGGCTAATTTTTTAGGGA,+1
21,BJPOL 3     ,GTAGGACCTACACCTGTCAAC,+21
21,SK69ENV     ,CTGTTGCAACTCACAGTCTGG,+21
21,SK33POL     ,CTGGTAGTCCAGGAATATGGC,+21
: 
22,BJGAG 1     ,TAGAAGAAATGATGACAGCATG,+1
22,ORENV 2     ,TCCTCAGGAGGGGACCCAGAAA,+22
22,SK104GAG    ,GACATAAGACAAGGACCAAAGG,+22
:
23,MSPOL 1     ,GGAATTGGAGGTTTTATCAAAGT,+1
23,MSENV 2     ,ACAGTACAATGTACACATGGAAT,+1
23,ORENV 1     ,CTGCTGTTAAATGGCAGTCTAGC,+1
:
24,BJGAG 2     ,ACAGGAGCAGATGATACAGTATTA,+24
24,BJPOL 1     ,ACAGGAGCAGATGATACAGTATTA,+1
:
25,BJPOL 2     ,CTGTACCAGTAAAATTAAAGCCAGG,+25
25,CO71ENV     ,TGTGGAGGGAATTCTTCTACTGTAA,+1
:
27,GAG5        ,GAGCATTCTGGACATAAGACAAGGACC,+27
:
28,SK38GAG     ,ATAATCCACCTATCCCAGTAGGAGAAAT,+1
28,SK39GAG     ,GCATTCTGGACATAAGACAAGGACCAAA,+28
:
29,87-29VIF    ,ACACCATATGTATGTTTCAGGGAAAGCTA,+1
:
31,CO72ENV     ,ATGAGGGACAATTGGAGAAGTGAATTATATA,+31
31,88-77VIF    ,CCTTATTAGGACACATAGTTAGCCCTGGTGT,+31