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Pregnancy Timeline by SemestersFetal liver is producing blood cellsHead may position into pelvisBrain convolutions beginFull TermWhite fat begins to be madeWhite fat begins to be madeHead may position into pelvisImmune system beginningImmune system beginningPeriod of rapid brain growthBrain convolutions beginLungs begin to produce surfactantSensory brain waves begin to activateSensory brain waves begin to activateInner Ear Bones HardenBone marrow starts making blood cellsBone marrow starts making blood cellsBrown fat surrounds lymphatic systemFetal sexual organs visibleFinger and toe prints appearFinger and toe prints appearHeartbeat can be detectedHeartbeat can be detectedBasic Brain Structure in PlaceThe Appearance of SomitesFirst Detectable Brain WavesA Four Chambered HeartBeginning Cerebral HemispheresFemale Reproductive SystemEnd of Embryonic PeriodEnd of Embryonic PeriodFirst Thin Layer of Skin AppearsThird TrimesterSecond TrimesterFirst TrimesterFertilizationDevelopmental Timeline
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Home | Pregnancy Timeline | News Alerts |News Archive Sep 19, 2013

 

Typically, it takes a skilled technician in a properly equipped laboratory 1-2 days to extract DNA from a sample, quantify the amount, make multiple copies of specific genetic sequences (PCR amplification), and then create a "DNA signature" that is unique to an individual.

In constrast,
GEMBE separates specific components of a sample by a molecular "tug-of-war."

The sample is pushed in one direction by an electric field and in the other by the counterflow of a buffer solution. Gradually reducing the buffer flow allows selected components from the sample to pass into a microfluidic channel to be analyzed. Unwanted components of the crude sample are kept out.


Laboratory technician pipettes dirt dissolved in a buffer solution from which DNA will be recovered using a novel NIST/Applied Research Associates microfluidic extraction technique. The dirt, originally collected on a swab (lower section seen in vial) like those on the right, is an example of a crude sample from which DNA for human identification has typically been difficult to obtain.

Credit: Michael E. Newman, NIST







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Microfluid technique recovers DNA for human IDs

Scientists have improved microfluid techniques for recovering DNA from real-world, complex mixtures such as dirt.

A team of researchers at the National Institute of Standards and Technology (NIST) and Applied Research Associates, Inc. (ARA, Alexandria, Va.) has demonstrated an improved microfluidic technique for recovering DNA from real-world, complex mixtures such as dirt.

According to a recent paper,* their technique delivers DNA from these crude samples with much less effort and in less time than conventional techniques. It yields DNA concentrations that are optimal for human identification procedures and can potentially be miniaturized for use outside the laboratory.

Forensic DNA testing is extensively used to link individuals to crimes, establish paternity, solve missing person cases, identify casualties in military and mass fatality events, and provide genealogical histories. Typically, it takes a skilled technician in a properly equipped laboratory 1-2 days to extract DNA from a sample, quantify the amount, make multiple copies of specific genetic sequences (PCR amplification), and then create a "DNA signature" that is unique to an individual.

However, when crude samples are the source of the desired DNA, the contaminants and particulates mixed in with the genetic material can seriously complicate the reading of a complete and accurate DNA signature. The additional purification steps needed for conventional means of handling crude samples, such as filtering, not only lengthen the processing time but also tend to reduce the quantity and concentration of DNA delivered—making human identification more difficult or impossible.


The new NIST/ARA technique is based on one the team first developed four years ago** for crude samples called "gradient elution moving boundary electrophoreses" or GEMBE. GEMBE separates specific components of a sample by a molecular "tug-of-war."

The sample is pushed in one direction by an electric field and in the other by the counterflow of a buffer solution. Gradually reducing the buffer flow allows selected components from the sample to pass into a microfluidic channel to be analyzed. Unwanted components of the crude sample are kept out.


To work with DNA, the researchers modified GEMBE so that two different buffer solutions—one with ions that move quickly and one with ions that move slowly during electrophoresis—are placed in the separate reservoirs connected by the microchannel. When a crude sample is suspended in the slow ion solution and electric current is applied, the DNA within the sample moves into the microchannel and concentrates at the interface between the two buffers.

Unwanted contaminants and particulates—including those that can inhibit PCR amplification—are left behind. The collected DNA can be quantified directly in the microchannel and then delivered into a vial for further processing.

To demonstrate the forensic capabilities of its new technique, the NIST/ARA team extracted, purified, quantified and concentrated human genomic DNA from both clean and dirty buccal (cheek cell) swabs. In both cases, the process yielded full DNA signatures.

The work is published in the journal Electrophoresis, September 2, 1013.

Abstract
Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.


* E.A. Strychalski, C. Konek, E.L.R. Butts, P.M. Vallone, A.C. Henry and D. Ross. DNA purification from crude samples for human identification using gradient elution isotachophoresis. Electrophoresis, Vol. 34, No.17, pp. 2522. Published online Sept. 2, 2013. DOI 10.1002/elps.201300133.

** http://www.nist.gov/public_affairs/tech-beat/tb20091117.cfm#gembe
and
http://www.nist.gov/public_affairs/tech-beat/tb20110830.cfm#gembe

Original press releas: http://www.eurekalert.org/pub_releases/2013-09/nios-djm091813.php