Michael J. Lane

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or tm of short duplex DNA oligomers (approximately 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting tm from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental tms of short duplex DNA oligomers is assessed.

MELTING STUDIES OF SHORT DNA HAIRPINS: INFLUENCE OF LOOP SEQUENCE AND ADJOINING BASE PAIR IDENTITY ON HAIRPIN THERMODYNAMIC STABILITY

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self-complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature-induced melting transitions in buffered 115 mM Na(+) solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5 degrees C and are concentration independent over strand concentrations of 0.5 to 260 microM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, DeltaH(cal), DeltaS(cal), and DeltaG(cal), were determined from DSC measurements. Model dependent transition parameters, DeltaH(vH), DeltaS(vH), and DeltaG(vH) were estimated from a vant Hoff (two-state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, DeltaH(cal) (DeltaH(vH)) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy DeltaS(cal) (DeltaS(vH)) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies DeltaG(cal) (DeltaG(vH)) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two-state model reveal that 10 of the 28 hairpins display non-two-state melting behavior. The database of sequence-dependent melting free energies obtained for the hairpins was employed to extract a set of n-n (nearest-neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest-neighbor loop sequence parameters.

Double-minute chromosomes as megabase cloning vehicles

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.

Variation in genomic Alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human:rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse:human hybrid cells that span the entire long arm of human chromosome 17.