SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem–loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.     

Self-formed adaptor PCR: a simple and efficient method for chromosome walking

We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb. SEFA PCR is simple and efficient and should have broad applications in the isolation of unknown sequences in complex genomes.     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

Slide track analysis of the relative motion between femoral head and acetabular cup in walking and in hip simulators

Joint simulators are important tools in wear studies of prosthetic joint materials. The type of motion in a joint simulator is crucial with respect to the wear produced. It is widely accepted that only multidirectional motion yields realistic wear for polyethylene acetabular cups. Multidirectionality, however, is a wide concept. The type of multidirectional motion varies considerably between simulators, which may explain the large differences in observed wear rates. At present, little is known about the relationship between the type of multidirectional motion and wear. One illustrative way to compare the motions of various hip simulators is to compute tracks made on the counterface by selected points of the surface of the femoral head and acetabular cup due to the cyclic relative motion. A new computation method, based on Euler angles, was developed, and used to compute slide tracks for the three-axis motion of the hip joint in walking, and for two hip simulators, the HUT-3 and the biaxial rocking motion. The slide track patterns resulting from the gait waveforms were found to be similar to those produced by the HUT-3 simulator. This paper is the first to include a verification of the computed simulator tracks. The tracks were verified in the two simulators using sharp pins, embedded in acetabular cups, engraving distinct grooves onto the femoral heads. The engravings were identical to the computed tracks. The results clearly differed from earlier computations by another research group. This study is intended to start a thorough investigation of the relationship between the type of multidirectional motion and wear.     

Achilles tendon loading during walking: application of a novel optic fiber technique

An optic fiber (? 0.5 mm) was utilized for the study of Achilles tendon forces (ATF) in eight volunteers who walked over a 10 m force platform at three speeds (1.1 ± 0.1 m × s⁻¹, 1.5 ± 0.1 m × s⁻¹ and 1.8 ± 0.2 m × s⁻¹). The presented ATF-time curves showed great intersubject variation in magnitudes of the sudden release of force after initial contact and in the peak ATF's (1430 ± 500 N). This intersubject variation in the peak force decreased only by 4% when cross-sectional area of the tendon was considered. Measured ground reaction forces and plantar pressures confirmed that the subjects walked quite normally during recordings. The peak ATF was found to be rather insensitive to speed in contrast to the rate of ATF development which increased 32% ( p < 0.5) from slow to fast walking speed. It is concluded that the optic fiber technique can be applied to study loading of the musculo-tendinous complex during normal locomotion such as walking. Accepted: 13 October 1997     

Nonlinear analysis of gait kinematics to track changes in oxygen consumption in prolonged load carriage walking: A pilot study

Linking human mechanical work to physiological work for the purpose of developing a model of physical fatigue is a complex problem that cannot be solved easily by conventional biomechanical analysis. The purpose of the study was to determine if two nonlinear analysis methods can address the fundamental issue of utilizing kinematic data to track oxygen consumption from a prolonged walking trial: we evaluated the effectiveness of dynamical systems and fractal analysis in this study. Further, we selected, oxygen consumption as a measure to represent the underlying physiological measure of fatigue. Three male US Army Soldier volunteers (means: 23.3 yr; 1.80 m; 77.3 kg) walked for 120 min at 1.34 m/s with a 40-kg load on a level treadmill. Gait kinematic data and oxygen consumption (VO₂) data were collected over the 120-min period. For the fractal analysis, utilizing stride interval data, we calculated fractal dimension. For the dynamical systems analysis, kinematic angle time series were used to estimate phase space warping based features at uniform time intervals: smooth orthogonal decomposition (SOD) was used to extract slowly time-varying trends from these features. Estimated fractal dimensions showed no apparent trend or correlation with independently measured VO₂. While inter-individual difference did exist in the VO₂ data, dominant SOD time trends tracked and correlated with the VO₂ for all volunteers. Thus, dynamical systems analysis using gait kinematics may be suitable to develop a model to predict physiologic fatigue based on biomechanical work.     

Acetone preservation: a practical technique for molecular analysis

In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.     

Female Acheta domesticus track acoustical and visual targets with different walking modes

ABSTRACT. Female Acheta domesticus L. were tested for tracking model calling songs with different syllable periods on the Kramer treadmill in the dark, under homogenous light, and in the presence of a visually attractive target (black square) positioned either perpendicular to or in front of the invisible acoustical target. The females exhibited tracking of the two targets by different walking modes.     

A numerical method for simulating the dynamics of human walking

This paper presents a general method for simulating the movement of the lower extremity during human walking. It is based upon two separate algorithms: one for single support (an open kinematic chain), and the other for the double support phase (a closed-loop linkage). Central to each of these is the recursive Newton-Euler inverse dynamics algorithm, applicable, as given, to any serial, spatial linkage.

For the unconstrained single support model, the Newton-Euler scheme is applied directly to numerically generate the equations of motion. In the case of double support, however, the kinematic constraint equations are used to first eliminate the redundant degrees of freedom, and then solve for the unknown ground reactions under the constrained limb.

The attractiveness of the method is that it offers a compact alternative to manually deriving the equations defining a mathematical model for human gait.     

A method for calculating the probability of tripping while walking

Despite tripping being one of the frequently reported causes of falls, currently there is no method of quantifying the probability of an individual's foot contacting obstacles during gait. This paper describes a statistical modeling technique based on variability in minimum toe clearance (MTC) data during treadmill walking to estimate the probability of the toe contacting an obstacle. MTC data were calculated from two foot markers and using a 2D geometric model of the distal end of the foot. Probability of tripping (PT) was calculated by modeling and then integrating the MTC sample distribution. Results from a young male subject continuously walking for 1 hour show the MTC distribution is not normally distributed with mean=1.03 cm, S.D.=0.25 cm, skew=1.01 and kurtosis=3.47. For this distribution, PT for an unseen 0.2 cm high obstacle is calculated to be 1 in every 10,363 strides. Without skew- and kurtosis-modeling PT reduced to 1 in every 1901 strides, which highlights the importance of skew and kurtosis-modeling for PT estimation. Predicted PT is seen to increase with increasing obstacle heights (e.g. PT for an unseen 0.5 cm obstacle is 1 in 95 strides and PT for an unseen 1.0 cm obstacle is 1 in 2 strides). The method presented in this paper shows that variability in MTC data can be modeled to quantify the probability/risk of tripping on obstructions/obstacles in the travel terrain, and has the potential for wide application in the areas of falls prediction and falls minimization.