The isolation of genomic DNA from blood typically involves digestion of nuclei with a combination of Proteinase K and SDS followed by deproteinization with organic reagents such as phenol and chloroform. Additional purification steps such as extensive dialysis, precipitation with a saturated solution of NaCl and/or absolute ethanol are then required for enzymatic analysis of the extracted DNA (1). The isolation procedure described here is both simple and rapid, eliminating the necessity for hazardous organic reagents. The method involves the incubation of nuclei with only Proteinase K at 65'C. It has been shown that Proteinase K is more active on denatured protein and that after prolonged incubation at 65°C it autoinactivates (2). As a result, following a 2 hour incubation, the extracted DNA can be used directly for enzymatic analysis without any additional purification. Nuclei were prepared by mixing an aliquot of whole blood with a 4 fold excess of ice cold CLB (0.32 M Sucrose, 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 1% Triton X-100). The lysate is centrifuged at 900 X g for 5 minutes at 4°C. The pellet is washed with CLB and following centrifugation the nuclear pellet is rinsed with a small aliquot of cold PLB (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 10 mM EDTA). For 1 ml of blood, the nuclei are resuspended in 250 ul of PLB+ ProK (PLB with 1 mg/ml Proteinase K) and incubated for 2 hours at 65°C. Periodic mixing during the 2 hour incubation is required for uniform lysis of the nuclear pellet and optimum recovery of DNA. A single 1.5 ml tube can be utilized for the entire procedure. The yield is greater than 90% of theoretical with an average size greater than 300 kb. DNA concentrations are determined spectrofluorometrically with Hoechst dye (3), or by electrophoresis with a series of known DNA samples on a 0.6% agarose gel stained with ethidium bromide. Conventional UV spectroscopy cannot be used since the proteolytic digestion products are not removed from the DNA. DNA isolated from blood is readily digestible with all restriction enzymes tested (Fig 1). The amount of restriction enzyme required for complete digestion is comparable with DNA purified by other methods. In addition to blood, DNA has been isolated from a variety of cell lines and tissues. The DNA is suitable for a number of other enzymatic modifications such as kinasing, ligation, and amplification by PCR. Utilization of this procedure allows the isolation of DNA from a large number of samples in less than 4 hours.
Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.
Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.The pollen grains of Tradescantia paludosa (16), corn (17), and tobacco (30) contain a store of presynthesized messenger RNAs (mRNAs) at the time of their release from the anther. These mRNAs have been shown to code in cell-free translation systems for proteins that are similar to the proteins synthesized during pollen germination and tube growth (9,17 shown that a large fraction (>64%) of the genes expressed in pollen are also expressed in vegetative tissues, whereas no more than 60% of the genes expressed in shoots are similar to those expressed in pollen (31). Similar hybridizations have been carried out with RNA from corn pollen and the results are similar to those obtained with Tradescantia (RP Willing, JP Mascarenhas, unpublished data). To further our knowledge of pollen development it is essential that the genes that are expressed in pollen, especially those unique to pollen, be isolated and characterized in some detail in order to understand their developmentally specific regulation and functions. We report here the construction of two cDNA libraries made to pollen mRNAs from Tradescantia and corn, and the utilization of some of the clones to answer questions about the nature of the pollen expressed genes and their transcription during pollen development and tube growth. MATERIALS AND METHODSPlant Material. Tradescantia paludosa L. plants were grown in the greenhouse and pollen was collected and stored as previously described (15). Corn (Zea mays L.) pollen was collected from field grown plants of the cultivar 'Gold Cup' (Harris Seeds, Rochester, NY). Pollen was quick frozen in liquid N2 and stored at -70°C. For later experiments the inbred line of corn W-22 (Illinois Foundation Seeds) was used. Various vegetative and ...
The attack on the World Trade Center on 9/11/2001 challenged current approaches to forensic DNA typing methods. The large number of victims and the extreme thermal and physical conditions of the site necessitated special approaches to the DNA-based identification. Because of these and many additional challenges, new procedures were created or modified from routine forensic protocols. This effort facilitated the identification of 1594 of the 2749 victims. In this Policy Forum, the authors, who were were members of the World Trade Center Kinship and Data Analysis Panel, review the lessons of the attack response from the perspective of DNA forensic identification and suggest policies and procedures for future mass disasters or large-scale terrorist attacks.
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