CCR5 Ligands in HIV Vaccines

transcriptase. After incubation at 370C 30', the cDNA is diluted into 200 l of a solution
containing 0.2mM each dNTP, 35mM Tris (pH 8.3), 50mM KC1, 7mM DTT, 2mM
MgCl2, and 50pmol of one of two oligonucleotide primers (5'TTCTTGGCTCTGCTGACACTCGAGC-3' for amplification of non-coding strands; 5'CCGATCACAGCCCTGAACAAAAGCA-3' for amplification of coding strands). This
mixture is then used for 35 cycles of PCR (940C for 1', 55C for 1', 720C for 2'.
Amplified products are then purified with PCR purification columns (Qiagen), and
digested with Taql or BstNI (New England Biolabs). LD78f-specific bands are
recognized as follows. TaqI digestion yields two bands of 362 and 328 bps when the
amplified gene is LD78J3, and three bands, of 325, 183, and 179 bp when MIP-lca is
amplified. BstNI digestion yields three bands of 394, 207, and 110 base pairs for the
amplified product of LD78f3, and two bands of 394 and 314 bps for the amplified product
of MIP-1a. Undigested and digested amplification products are run on a 4% NuSieve
GTG gel. The gel is then stained with ethidium bromide and scanned an Alpha Imager
2000 apparatus (Alpha Innotech, CA) and the intensity of the two bands will be measured
by densitrometic reading. We will then calculate the ratio of LD78 f3/MIP1 a abundance.
Alternative approaches. We have successfully used for our cross-sectional study
commercially available RANTES, MIP- la, and MIP-1f3 ELISAs, and found them to be
very sensitive, reliable, and reproducible Therefore, we do not expect any particular
problem with these ELISAs, although our ELISA core facility will regularly check their
performance to verify both sensitivity and reproducibility. Similarly, we do not expect to
incur into any particular problem with
our own -2 RANTES ELISA. Should
we incur in problems in discriminating
NASBA Amplification Reaction and quantifying LD78j3 and MIP-loa -
Legend: Prerspecific cellular RNAs, we will adopt a
ansense RNA ves racpts
sense eRNARe tdifferent protocol, based on nucleic
* sense DNA a
Sansense DNA-RNaH acid sequence-based amplification
Re____ Tr_ nptr e (NASBA). NASBA (47) is actually one
component of a total technology system
PrmerRNpdhnese which also includes a versatile nucleic
Rse n. r Rever Treciptacid isolation procedure (48), and a
Hw g -r1powerful detection technology
Figure 1: The NASBA Amplifcation Pathway involving electrochemiluminescence
(ECL) (49). The Boom extraction
Figure 1. Schematic representation of the method can be applied to a wide range
of sample types including whole blood,
plasma, serum, cells, tissue, sputum,
feces, and cerebro-spinal fluid. The ECL technology allows for the sensitive detection of
amplification products in both qualitative and quantitative configurations.
Electrochemiluminescence (ECL) technology has been adapted for use with NASBA
technology in a quantitative format, through the utilization of an internal standard (50).
NASBA amplification can be applied to both RNA and DNA targets. The reaction
process for RINA analytes is depicted in Figure 1.