Biomedical subjects

M M Darfler

Publications and source records attributed to M M Darfler.

3 recordsLinked to original sources

PubMed2005

Printing of protein microarrays via a capillary-free fluid jetting mechanism.

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.

Alkaline Phosphatase↗

PubMed1999

Long PCR for VNTR analysis.

The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.

Amino Acid Sequence↗

PubMed1991

Occurrence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction.

More than 22 types of human papillomavirus (HPV) have been detected in genital tract squamous cell intraepithelial lesions. Seven of two hundred eighty-six (2.4%) genital tract tissues in which HPV DNA was detected by in situ hybridization contained two or more different HPV types. When analyzed by site, 5 of 204 (2.4%) of cervical intraepithelial lesions were infected by more than one type, compared with 2 of 82 (2.4%) of vulvar lesions. The rate for low-grade lesions was similar (5/218; 2.3%) to that for high-grade lesions (2/68; 2.9%). In contrast, two different HPV types were detected in 6/33 (18%) of tissues by the polymerase chain reaction (PCR) using type-specific primers for eight HPV types. It is concluded that infection by one HPV type is rarely associated with concurrent 'active' infection by a second HPV type, even though DNA of a different viral type can be detected by PCR in about one fifth of such cases. Further study is required to determine if an existing HPV infection can inhibit replication by a different HPV type.

DNA, Viral↗