Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq.

Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2×250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq.

Can autism be diagnosed accurately in children under 3 years?

This study investigated the reliability and stability of an autism diagnosis in children under 3 years of age who received independent diagnostic evaluations from two clinicians during two consecutive yearly evaluations. Strong evidence for the reliability and stability of the diagnosis was obtained. Diagnostic agreement between clinicians was higher for the broader discrimination of autism spectrum vs. no autism spectrum than for the more specific discrimination of autism vs. PDD-NOS. The diagnosis of autism at age 2 was more stable than the diagnosis of PDD-NOS at the same age. Social deficits and delays in spoken language were the most prominent DSM-IV characteristics evidenced by very young children with autism.