From: msalminen@nphi.fi
Newsgroups: bionet.molbio.hiv
Subject: HIV PCR primers
Date: 16 Apr 92 11:30:47 GMT
Organization: National Public Health Institute, Finland

Hello Retrovirologists !

As i have noticed that the discussion seems to be a bit jammed on this forum i
think it is time for me to bring up some questions for discussion concerning
the research on the evolution of HIV.

As many of you surely know, HIV is one of the fastest evolving "organisms"
known. It's rate of primary mutations is of the order of 10-3 misincorporations
per base. The fixing rate is about 10-4 so it aqcuires at least one base
substitution per replication of the genome. Additionally, insertions/deletions
and recombination is frequent.

This leads to many different antigenic forms of the virus. In fact all isolates
from around the world are different antigenetically. This is a major obstacle
in the design and development of vaccine candidates, especially as the primary
neutralizing epitope of HIV (thought to be important for protection) is one of
the most variable sites and changes very fast during the infection also.

One way of coping with this (not the only possibility though) is to try to make
out the amount of variation and the possible limits of this, and to design a
vaccine that would contain as many variants of common antigenic types as
possible, hoping that such a vaccine would be sufficiently cross-protecting to
be able to stop the infection. I know that i am simplifying things but the
basic idea is about like this.

This kind of work is actually on it's way already, there are WHO, NIH/CDC and
EC cooperations going on. The project, which is global, is called

		INTERNATIONAL HIV TYPING NETWORK
			   (IHTN)  

and is part of the WHO technical working group on HIV isolation and
characterization. This effort is aimed at the collection and characterization
of viral variants around the world, starting in Africa. Part of the work will
be the sequencing of many envelope sequence variants especially from the V3
region of the gene.

This approach is aimed at the collection of clones for possible candidate
vaccines and for the elucidation of the actual amount of variation in natural
isolates over the world.

A crucial factor in this approach is the development of PCR primers. As a
consequence of the variation it is _very_ difficult to design truely universal
primers for env and the other genes that would amplify all natural isolates.

Many labs over the world have been designing PCR primers for the detection of
HIV with various success. It would be of advantage for the HIV research
community to have the primer sequence and the amplification conditions gathered
in a database that would be in the public domain and freely distributed. I
propose the establisment of such a database. This could function so that the
entries would be sent to this newsgroup and i would collect them from the group
and append to the database which would then be deposited in various ftp
archiwes such as nic.funet.fi and the embl mail netserver. I could start the
database by sending out entries of my own PCR primers and a collection from the
litterature.

Below is a suggestion for a database entry form. Please pass this suggestion
along to other HIV-researchers and comment on the database entry form.
Discussion about a database management environment is also welcome. I could put
the sequences into Seqaid site format and possibly Genbank and GCG also. I
think it would be an advantage to have the sequences in a different file from
the other information with a pointer to it (i.e. MSGAG1.SEQ and MSGAG1.TXT). 

P.S. Remember that the PCR patent is now owned by ROCHE and only those Taq
polymerases and kits that are licenced by them may be used for PCR.

	Hoping for vigorous discussion and many suggestions,

	Mika Salminen, HIV-Unit, FINNPHI Helsinki
	msalminen@nphi.fi (In)
	msalminen@finnphi (Bitn)

Disclaimer: I have no connection to Perkin-Elmer/Cetus, Roche, or any
	    commercial company whatsoever

Suggested database entry form:

sequence file: MSGAG1.SEQ
______________________ cut here _______________________________________________
>MSGAG1						(primername)
ACGTACGTACGTACGTACGT                 		(<-sequence + strand)
______________________ cut here _______________________________________________

Text File: MSGAG1.TXT
______________________ cut here _______________________________________________
PRIMER:		MSGAG1				(pointer to sequence file)
REF: 		M.Salminen et al. (1975).	(reference if published)
		J.Irr.Res. 0; 111-1111	
GENE:		GAG				(gene(s))
LOCUS: 		1111-1133			(in HXB2R reference)
5'-END: 	+	    			(+ or - strand primer)
COMBINATIONS: 	MSGAG2,MSGAG3,MSGAG4  		(TESTED amplifications)
BEST:		MSGAG2                          (BEST combination)
PRODUCTS:	234,345,456             	(amplification product length)
NESTED:		Y				(Y if needed otherwise N)
OUTER:		MSGAG0+MSGAG3,MSGAG1+MSGAG4     (if needed in primary PCR)
BEST:           MSGAG0+MSGAG3                   (BEST combination)
INNER:		NONE				(if can be used as outer)
BEST:		NONE                            (BEST combination)
TEMPLATE:	PBL:DNA,LYSATE 			(Quality of template tested)
PROFILE:	95,30                           (temp,time in sec.)
		55,30
		72,180
CYCLES:		25                              (cycles)
BUFFER:		10mm TRIS,50mm KCl,1.5mm MgCl   (PCR buffer)
PRIMER:		1 uM				(Primer conc.)		
dNTP:		200 uM                          (dNTP concentration)
TAQ:		2.5 u                           (Units of Taq)
VOLUME:		50 ul                           (Volume of PCR)
EFFICIENCY:	AM/EUR:95,12345,		(Sample origin:detection rate%
		AFR:85,123			 for BEST, samp.tested)
		ASIA:90,1234