4,415,732 11/1983 Caruthers et al..................... 536/27 4,458,066 7/1984 Caruthers et al. ... . 536/27 4,500,707 2/1985 Caruthers et al. ... 536/27 4,668,777 5/1987 Caruthers et al. .................... 536/27 4,762,779 8/1988 Smitman .................................. 436/6 4,771,384 9/1988 Daniels et al., ........................ 935/75 4,965,349 10/1990 Woo et al. ............................ 536/27

The conditions for hybridization and detection of enzyme-labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme-linked probe which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity that observed for 32P-labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi-Phos Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.

The single-locus probe LH1 (D5S110) originally described by Armour, J.P., et al, Genomics, Vol. 8, p.501, 1990 was evaluated for its effectiveness in DNA typing assays for human identification. A cloned, purified, 6-Kb DNA fragment containing sequences complementary to the D5S110 locus was labeled with 32P-dCTP by the random primer method. LH1 appears to be one of the most sensitive single-locus probes observed, capable of detecting a DNA profile in 10 ng of Hae III restricted genomic DNA in a 24-hour autoradiographic exposure. An enzyme-linked oligonucleotide probe was constructed for use in a chemiluminescent hybridization and detection format. Nine repeats within the D5S110 cloned insert were sequenced, and a 30-base oligonucleotide was synthesized and labeled with alkaline phosphatase. The sensitivity and specificity of the alkaline phosphatase labeled oligonucleotide under the ACES 2.0 hybridization and detection conditions is comparable to that of the 32P labeled LH1 probe. The enzyme-linked oligonucleotide probe was capable of detecting the correct banding pattern in 25 ng of Hae III restricted genomic DNA in a 3-hour exposure following a 3-hour ramp.

There are two basic DNA typing methodologies available to the forensic scientist for characterizing biological evidence. The first technology to gain wide use in the forensic arena was typing of DNA for variable number of tandem repeat (VNTR) loci by restriction fragment length polymorphism (RFLP) analysis. RFLP typing is well-defined, provides a high degree of discrimination, and can be accomplished, at times, with less than 50 ng of high molecular weight genomic DNA. This methodology has been validated for forensic applications (Budowle and Baechtel 1990). The other strategy for typing DNA is based on increasing the number of copies of a target sequence of DNA by amplification using the polymerase chain reaction (PCR) (Saiki, et al. 1985). Since the number of target sequences of interest is increased dramatically by PCR, simplified typing methods can be used for determining DNA polymorphisms in a sample. The advantages a PCR-based technology offers, compared with the currently employed RFLP approach, are: 1) augmented sensitivity and specificity, 2) decreased assay time and labor, 3) absence of an isotopic label, and 4) many degraded DNA samples can be amplified by PCR and subsequently typed because alleles amenable to PCR are much smaller in size compared with alleles detected by RFLP analysis. These qualities combine to make PCRbased technologies extremely useful tools for analyzing biological material found at crime scenes.

The sensitivity, selectivity, and ease of use of nucleic acid probes and the availability of efficient protocols for the isolation and cloning of specific DNA sequences have led to the development of a wide selection of probes for biomedical and clinical applications. In the basic hybridization protocol, a labeled nucleic acid probe is annealed to a complementary DNA or RNA target sequence, which either is in solution or is immobilized on an inert support. The labeled nucleic acid probe is used to determine the presence or absence of the target sequence in the reaction mixture. An effective nonradioactive detection system should recognize the annealed nucleic acid probe with a degree of precision comparable to that obtained in the primary hybridization reaction. The presence of either a free amine or biotin may also be ascertained by chemical analysis. Compounds containing primary amines give positive color development when spotted on thin layer chromatography (TLC) plates and sprayed with ninhydrin. The first approach uses iminobiotin, an analog of biotin in which the ureido group has been replaced with a guanido group. Iminobiotin binds to avidin with a binding constant that increases with increasing pH and nonprotonated iminobiotin binds efficiently to avidin.

We have synthesized and analyzed the functional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.

A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA. Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.

The identification of left handed or Z-DNA in solutions of poly d(GC) in high salt suggests that left handed DNA may exist in biological systems if stabilized at lower ionic strength. In the present study we show that binding of polyarginine to the Z form of poly d(GC) results in a protein-Z-DNA complex stable near physiological ionic strength. The percentage of Z-DNA in the low salt polyarginine-poly d(GC) complex was measured from the DNA circular dichroism spectrum. The ratio of Z to B-DNA is a linear function of polyarginine concentration and is sensitive to proteolytic digestion by trypsin. These results suggest that arginine-rich proteins may stabilize Z-DNA in vivo.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.

High-resolution nmr of exchangeable protons in the side chain of arginine reveals two distinct resonances arising from restricted rotation about the N(1)–C(ε) bond. Spectral assignments based upon pH-dependent proton-exchange behaviour identified each resonance as arising from one of the magnetically distinct guanidinium amino groups in the molecule. Computer simulation of the temperature-dependent coalescence of these peaks defines an activation energy of 14.3 kcal/mol for internal rotation about this bond. Similar results to those observed in monoarginine are reported for diarginine, triarginine, and the arginine-rich histone tetramer. Based on these findings, a nonsymmetric mode of arginine–ligand interaction is suggested, and the molecular dynamics of proton exchange in the arginine side chain is discussed.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.

We report transient electric dichroism experiments on nucleosomal core particles containing 140 and 175 base pairs of DNA, and on spacerless dinucleosomes. The results indicate that all particles posses a permanent dipole moment. The orientation time of 140 base pair nucleosomes implies an estimated maximum dimension of a = 130 A (a must be at least 111 A), consistent with the disk model. The maximum dimension of the spacerless dinucleosome is estimated to be about 290 A (at least 180 A), ruling out a structure in which two disks are stacked directly on top of each other. The reduced dichroism amplitude indicate that the DNA superhelix axis in nucleosomes aligns perpendicular to the electric field, as expected for a dipole moment directed along a C2 symmetry axis across the disk diameter. Nucleosomes containing 175 base pairs of DNA show a substantially larger dichroism amplitude that do 140 base pair nucleosomes. In the context of the disk model, this result is shown to be consistent with 100 base pairs of DNA per superhelical turn, but not with 80 base pairs per turn.

Biochemical, spectroscopic, and hydrodynamic studies were performed on the reconstituted complex of 140 base pair DNA and the arginine-rich histone tetramer (H3/H4)2. The histones bind to DNA in a 1:1 molar ration to form a stable particle which orients in an electric field with a rotational correlation time of 6.3 mus and a limitign reduced dichroism of --0.74. The complex was modeled hydrodynamically as a cylinder of dimensions 450 X 80 X 80 A containing approximately 1.5 superhelical turns. Addition of the lysinerich histones to this complex cause a condensation of the structure and results in physical properties nearly identical with those of a native nucleosomal particle.