Category Archives: DNA

Schematic of single molecule DNA sequencing by a nanopore with phosphate-tagged nucleotides. Each of the four nucleotides will carry a different tag. During SBS, these tags, attached via the terminal-phosphate of the nucleotide, will be released into the nanopore one at a time where they will produce unique current blockade signatures for sequence determination. A large array of such nanopores will lead to high throughput DNA sequencing.

(Phys.org)DNA sequencing is the driving force behind key discoveries in medicine and biology. For instance, the complete sequence of an individual's genome provides important markers and guidelines for medical diagnostics and healthcare. Up to now, the major roadblock has been the cost and speed of obtaining highly accurate DNA sequences. While numerous advances have been made in the last 10 years, most current high-throughput sequencing instruments depend on optical techniques for the detection of the four building blocks of DNA: A, C, G and T. To further advance the measurement capability, electronic DNA sequencing of an ensemble of DNA templates has also been developed.

Recently, it has been shown that DNA can be threaded through protein nanoscale pores under an applied electric current to produce electronic signals at single molecule level. However, because the four nucleotides are very similar in their chemical structures, they cannot easily be distinguished using this technique. Thus, the research and development of a single-molecule electronic DNA sequencing platform is the most active area of investigation and has the potential to produce a hand-held DNA sequencer capable of deciphering the genome for personalized medicine and basic biomedical research.

A team of researchers at Columbia University, headed by Dr. Jingyue Ju (the Samuel Ruben-Peter G. Viele Professor of Engineering, Professor of Chemical Engineering and Pharmacology, Director of the Center for Genome Technology and Biomolecular Engineering), with colleagues at the National Institute of Standards and Technology (NIST) led by Dr. John Kasianowicz (Fellow of the American Physical Society), have developed a novel approach to potentially sequence DNA in nanopores electronically at single molecule level with single-base resolution. This work, entitled "PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis" is now available in the open access online journal, Scientific Reports, from the Nature Publication group.

The reported nanopore-based sequencing by synthesis (Nano-SBS) strategy can accurately distinguish four DNA bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides at the single molecule level for sequence determination. The basic principle of the Nano-SBS strategy is described as follows. As each nucleotide analog is incorporated into the growing DNA strand during the polymerase reaction, its tag is released by phosphodiester bond formation. The tags will enter a nanopore in the order of their release, producing unique ionic current blockade signatures due to their distinct chemical structures, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof-of-principle, the research team attached four different length polymer tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate (a modified DNA building block) and demonstrated efficient incorporation of the nucleotide analogs during the polymerase reaction, as well as better than baseline discrimination among the four tags at single molecule level based on their nanopore ionic current blockade signatures. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

In previous work, the Center of Genome Technology & Biomolecular Engineering at Columbia University, led by Professor Ju and Dr. Nicholas J. Turro (William P. Schweitzer Professor of Chemistry), developed a four-color DNA sequencing by synthesis (SBS) platform using cleavable fluorescent nucleotide reversible terminators (NRT), which is licensed to Intelligent Bio-Systems, Inc., a QIAGEN company. SBS with cleavable fluorescent NRTs is the dominant approach used in the next generation DNA sequencing systems. Dr. Kasianowicz and his group at NIST pioneered the investigation of nanopores for single molecule analysis. They previously reported that different length polymers, polyethylene glycols (PEGs), could be distinguished by their unique effects on current readings in a -hemolysin protein nanopores at single molecule level and subsequently developed a theory for the method. Their results provide the proof-of-concept for single molecule mass spectrometry. The combination of the SBS concept with the distinct nanopore-detectable electronic tags to label DNA building blocks led to the development of the single-molecule electronic Nano-SBS approach described the current Scientific Reports article.

As lead author Dr. Shiv Kumar points out, "The novelty of our approach lies in the design and use of four differently tagged nucleotides, which upon incorporation by DNA polymerase, release four different size tags that are distinguished from each other at the single molecule level when they pass through the nanopore. This approach overcomes any constraints imposed by the small differences among the four nucleotides, a challenge which most nanopore sequencing methods have faced for decades." Moreover, the technique is quite flexible; with PEG tags as prototypes, other chemical tags can be chosen to provide optimal separation in different nanopore systems.

With further development of this Nano-SBS approach, such as the use of large arrays of protein or solid nanopores, this system has the potential to accurately sequence an entire human genome rapidly and at low cost, thereby enabling it to be used in routine medical diagnoses.

More information: Scientific Reports, 2, 684 DOI:10.1038/srep00684, 2012

Journal reference: Scientific Reports

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Researchers report novel approach for single molecule electronic DNA sequencing

A Middlesex Superior Court judge is allowing two samples of DNA to be used as evidence in the trial of a Framingham man accused of murdering a couple in Hudson in 2010.

Judge Sandra Hamlin denied defense attorney Thomas Fords request to dismiss a sample of Velezs DNA that was found underneath Trisha Bennetts fingernail and a blood spatter found on the jeans Velez wore the night of the murders, said Stephanie Chelf Guyotte, a spokeswoman for the Middlesex District Attorneys office.

During a pre-trial conference earlier this week, Ford argued that a report did not note which portion of Bennetts fingernail the DNA sample was taken from. Ford said DNA can be transmitted to the top of another persons fingernail through casual contact. However, DNA is normally transmitted underneath another persons fingernail if there is sexual or defensive contact.

Ford also expressed concerns that there was no defense expert present at the swabbing and testing of the DNA.

Assistant District Attorney Joseph Gentile said testimony at a previous hearing documented the DNA sample was found underneath Bennetts fingernail.

In the case of the blood spatter on Velezs jeans, Ford said a report did not identify which blood spot was extracted and tested for DNA. The number of spots tested was also not in the report, said Ford.

Citing a report, Gentile said the sample was taken from a defined section near the left thigh Velezs jeans near his thigh.

Jury selection began Friday and will continue on Monday, said Guyotte.

Velez, 29, is charged with first-degree murder in the stabbing deaths of Bennett, 20, and her boyfriend Angel Ortiz, 23. Bennett and Ortiz were found dead inside their Emerson Gardens condominium May 1, 2010.

Authorities arrested Velez nearly three months after he called 911 in the early morning of May 1, 2010, telling police he and two friends had been stabbed. Police found Velez, who was suffering from stab wounds, in the parking lot. Prosecutors say Velezs wounds were self-inflicted.

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Judge denies motions to dismiss DNA evidence in Hudson murder case

It's history being made -- the FBI just this month took acceptance of its first-ever "Rapid DNA" equipment for near-instant DNA analysis in the field. But use of this DNA analysis-in-a-box, which can be carried around and connected to the Internet, may be slowed because current law never envisioned such analysis being done for law-enforcement purposes outside an accredited lab.

RELATED: FBI eager to embrace 'Rapid DNA' testing

That realization, brought to light at the Biometric Consortium Conference on Wednesday, cast a shadow on what's a shining moment for the biometrics industry and its partnership with the FBI. The FBI has spent years working to build Rapid DNA equipment according to careful designs for ruggedness, security and usefulness in generating individual DNA profile data that police stations could use to share and match against the FBI's existing DNA Index System (NDIS) database. Such Rapid DNA gear can take in a cotton swab of an individual's saliva or blood in the field and within about 90 minutes, automatically spit out a human DNA profile.

Dr. Thomas Callaghan , senior biometric scientist in the biometric analysis section of the FBI Laboratory, just this month took delivery on the first two working models of Rapid DNA machines, the RapidHit 200 made by integenX, and the ANDE box made by NetBio. "It really is a remarkable achievement," says Callaghan. He and many others in the biometrics field this week at the conference recognized the historic significance of the technology breakthrough presented by the first commercially-viable equipment for Rapid DNA.

The U.S. Army has started evaluation of two ANDE System boxes it got from NetBio, says Jeff Salyards, chief scientist at the U.S. Army Criminal Investigative Laboratory. He reports that the Rapid DNA technology supplied by ANDE appears to work effectively.

Richard Selden, CEO of NetBio, assures that the ANDE System boxes for DNA analysis have undergone military-standard testing for ruggedness. However, Salyards says more testing is needed, and cautioned military buyers, eager to use Rapid DNA equipment in the field, to show patience as more testing is done.

It also could be a while until Rapid DNA can be used for U.S. law enforcement purposes. The National Institute of Standards and Technology (NIST), which is teaming with the FBI to test the NetBio and IntegenX systems, as well as possibly others, for use with law enforcement, expects a full evaluation that includes new processes to be followed to connect to federal databases. Such an evaluation could take upwards of a year.

What's more, the DNA Identification Act of 1994 passed by Congress gave the FBI the authority to establish its DNA index system, but didn't envision that DNA information would be uploaded to the FBI database from a police station using Internet-connected Rapid DNA equipment. The law covers only accredited DNA labs in use today, not the mobile Rapid DNA equipment that can be operated by non-technical personnel anywhere, according to Clark Jaw, an auditor at the FBI Laboratory for the Combined DNA Index System (CODIS). It appears there needs to be a change to the DNA Identification Act to accommodate use of the new technology, he says.

Other obstacles to achieve full-scale use in law enforcement include the need to build out CODIS software to accommodate Rapid DNA and create a quality-assurance process system. That all means Rapid DNA for law-enforcement purposes in the U.S. may take time. But the first Rapid DNA equipment is known to already be in use among secretive intelligence agencies.

"The ultimate goal is to have that technology available for law enforcement use at the police station," Jaw says, pointing out that one day law enforcement officials should be able to carry out real-time DNA-related searches using the Rapid DNA equipment to aid in fast investigation of crime suspects and crime scenes.

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Legal hurdles threaten to slow FBI's 'Rapid DNA' revolution

Using DNA matches off a knife, San Luis Obispo police detectives were able to identify a suspect in an attempted murder case, and then arrested a Vallejo man for the alleged crime.

In a news release issued Thursday, police Lt. Jeff Smith said Austin Sarna, 21, was arrested Wednesday in Vallejo after the DNA evidence linked him to an attack that occurred in January.

Sarna was taken into custody without incident, and was transported to San Luis Obispo County Jail, where he remains on a no-bail warrant.

Smith said that at 12:30 a.m. on Jan. 20, police were called to the intersection of Broad and Monterey streets on the report of a man who had been stabbed and was bleeding profusely from his arm.

Police learned that several men got into an altercation with two or more others. Trevor Tice, 27, of Atascadero, was stabbed multiple times in the back and arm. Zachary Lerno, 26, was stabbed once in the head.

"During the investigation there were limited details regarding the suspect due to a lack of witnesses and levels of intoxication," Smith said. But the knife used in the attack was found by officers and taken as evidence.

The suspected weapon was sent to a state crime lab for analysis. One DNA sample matched Tice; another matched Sarna. Investigators then contacted the two male victims and witnesses, who identified Sarna as one of the men in the altercation.

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DNA evidence links Vallejo man to January stabbing in SLO, police say

George Zimmerman claims self defense in the killing of Trayvon Martin. But there's no DNA evidence that Trayvon Martin ever touched George Zimmerman's gun.

Forensic tests made public Wednesday show that George Zimmerman's was the only DNA that could be identified on the grip of the gun used to fatally shoot 17-year-old Trayvon Martin.

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The results rule out Martin's DNA from being on the gun's grip. Zimmerman's DNA also was identified on the gun's holster, but no determination could be made as to whether Martin's DNA was on the gun's holster, according to the report from the Florida Department of Law Enforcement.

Zimmerman is charged with second-degree murder for fatally shooting Martin during a confrontation in a gated community in Sanford in February. Zimmerman is pleading not guilty, claiming self-defense.

A delay in Zimmerman's arrest led to nationwide protests.

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The question of whose DNA is on the gun and holster could play a role in Zimmerman's defense.

Zimmerman says Martin had been on top of him, slamming his head against the ground and smothering his mouth and nose with his hand and arm when he grabbed his gun from a holster on his waist before Martin could get it. He shot the teenager once in the chest.

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George Zimmerman: No DNA evidence of a struggle for his gun

Only DNA from George Zimmerman was found on the grip of the gun that was used in the fatal shooting of unarmed teenager Trayvon Martin, according to DNA test results released Wednesday.

The test results were in the latest batch of discovery material released by officials in the notorious murder case, which has become a national flashpoint in race relations and gun laws. Zimmerman, 28, is free on bail awaiting trial on charges of second-degree murder in the killing of Martin, an African American teenager, who was shot to death in a confrontation at a gated community in Sanford, Fla., on Feb. 26.

The tests on the gun and the holster were performed by the Florida Department of Law Enforcement. They found Zimmermans DNA on the gun but could not find any of Martin's. No determination could be made as to whether Martin's DNA was on the holster.

PHOTOS: Tense moments from Trayvon Martin case

Zimmerman has acknowledged shooting Martin, but insists he acted in self-defense in the wake of a confrontation that left him injured.

The DNA findings could play a role in the trial, allowing the prosecution to argue against self-defense since it now seems that Martin never touched the weapon. The defense could counter that Zimmerman shot before Martin could get to the gun.

Zimmerman was not arrested the night of the shooting. After weeks of protests about that fact, a special prosecutor charged Zimmerman, a neighborhood watch volunteer, with second-degree murder.

Among other material released Wednesday was an interview with a clerk of the convenience store where Martin had gone to buy a package of Skittles and a can of iced tea. Martin was returning from the store when the confrontation with Zimmerman took place. The unidentified clerk told authorities he had no recollection of serving Martin.

To be honest, I don't even remember that day, he said.

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George Zimmerman's DNA, not Trayvon Martin's, found on gun

MADISON Anyone arrested for a felony offense and all adults convicted of misdemeanor crimes would be required to provide a DNA sample to law enforcement under a proposal from the state Department of Justice.

Adults arrested for certain misdemeanor crimes such as fourth-degree sexual assault and prostitution also would have to provide DNA samples, which would be entered into a national database used to match DNA evidence collected at crime scenes to suspects.

Currently, state law allows DNA to be collected only from adults and juveniles convicted of felonies, with about 12,000 samples obtained each year.

Brian OKeefe, administrator for the DOJs Division of Law Enforcement Services, said the expected addition of about 68,000 DNA samples a year at least initially under the proposed expansion would help law enforcement solve more cases more quickly and get criminals off the street.

The number of new samples would eventually drop, he said, because DNA profiles of those reoffending would not have to be added to the system.

But Chris Ahmuty of the ACLU of Wisconsin, said, It seems like theyve gone for the nuclear option when it comes to DNA on arrest.

Casting such a wide net raises concerns about cost, management and privacy and turns the presumption of innocence on its head, Ahmuty said. He added that the DOJ proposal is more encompassing and costly than previous legislative proposals.

OKeefe said the proposed expansion of DNA collection would begin in October 2014 and cost about $7 million in its first two years.

To cover those costs including the addition of 26 full-time positions DOJ is asking that everyone convicted of a felony be required to pay a $250 surcharge, and that everyone convicted of a misdemeanor pay a $150 surcharge.

Currently, a DNA surcharge for people convicted of felonies is often waived by a judge.

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Expansion of criminal DNA collection proposed

ScienceDaily (Sep. 18, 2012) Human papillomavirus (HPV) DNA positivity alone, particularly when assessed using polymerase chain reaction methods, is a poor biomarker for HPV-driven head and neck cancers, according to two studies published in Cancer Research, a journal of the American Association for Cancer Research. These studies identified alternative potential markers including viral load, viral gene expression and the evaluation of HPV DNA in combination with certain HPV assays.

Prior research has established that HPV is a cause of some head and neck cancers, including oropharyngeal cancer, and that patients with HPV-associated disease tend to have a better clinical outcome. Consequently, the proper assessment of the clinical status of individual tumors has become a goal of clinicians treating this disease because HPV at the tumor site does not indicate causal involvement in the cancer.

In the first study, Dana Holzinger, Ph.D., of the division of genome modifications and carcinogenesis at the German Cancer Research Center in Heidelberg, Germany, and colleagues analyzed the potential of direct and indirect HPV markers to identify patients with HPV-driven tumors.

They analyzed 199 oropharyngeal squamous cell carcinoma specimens for HPV DNA, viral load, RNA expression patterns seen in cervical carcinomas and the p16 protein, which is associated with tumor suppression.

Results indicated that the cervical cancer RNA expression pattern and viral load were associated with the lowest risk for death from oropharyngeal cancer. In contrast, a weaker association was found for samples that were HPV DNA-positive or that expressed the p16 protein.

"We showed that high viral load and a cancer-specific pattern of viral gene expression are most suited to identify patients with HPV-driven tumors among patients with oropharyngeal cancer," Holzinger said. "Viral expression pattern is a completely new marker in this field and viral load has hardly been analyzed before."

In a second study, researchers evaluated several biomarkers individually and in combination for overall survival among head and neck cancers including polymerase chain reaction-based and serological HPV DNA testing, and p16 immunohistochemistry.

They found that the expression of two oncoproteins, E6 and E7, was associated with improved survival in oropharyngeal disease. In addition, HPV DNA positivity or p16 expression combined with E6 and E7 expression were also associated with enhanced survival. However, neither HPV DNA positivity nor expression of p16 alone yielded a similar result.

"Assessment of HPV DNA using polymerase chain reaction methods as a biomarker in individual head and neck cancers is a poor predictor of outcome and is also poorly associated with antibody response indicative of exposure and/or infection by HPV," said study author Karl T. Kelsey, M.D., professor in the department of epidemiology and the department of pathology and laboratory medicine at Brown University in Providence, R.I. "We may not be diagnosing these tumors as accurately and precisely as we need to for adjusting treatments."

The next step in this research is further validating the findings of these two studies using head-to-head comparisons and developing assays for direct clinical application of the markers.

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Assessment of HPV DNA Alone Insufficient to Identify HPV-Driven Head and Neck Cancers

It's been the FBI's dream for years -- to do near-instant DNA analysis using mobile equipment in the field -- and now "Rapid DNA" gear is finally here.

The idea is that you simply drop into the system a cotton swab with a person's saliva, for example, and the "Rapid DNA" machine spits out the type of DNA data that's needed to pin down identity. Now that such equipment exists, the FBI is pushing to get it into the hands of law enforcement agencies as soon as possible. [Also see: "FBI building system that blows away fingerprinting"]

"DNA has emerged as the gold standard in forensics analysis," Steven Martinez, executive assistant director of the science and technology branch at the FBI, said in his keynote address to attendees of the Biometric Consortium Conference in Tampa on Tuesday.

RELATED: Biometrics scares people

Though the genetic information contained in an individual's DNA, which is in all human cells, has been used since the late 1980s to solve crime cases, analysis of DNA has remained frustratingly slow because DNA had to be sent to special labs to be analyzed. New "Rapid DNA" devices are now ready to be evaluated and the FBI has received two basic types.

One is called the RapidHIT, which is made by IntegenX, a Pleasanton, Calif.-based company whose CEO Stevan Jovanovich was in the exhibit hall to explain how the Rapid DNA device can spit out an individual's DNA data within 90 minutes.

Another company, NetBio, is also believed to have delivered its Rapid DNA-type equipment to the FBI, Jovanovich says, and the National Institute of Standards and Technology (NIST) is expected to play an important role in helping certify systems and processes for how these boxes will be used by the FBI and local police stations to collect DNA data on suspects.

Jovanovich notes that the networked IntegenX RapidHIT box, which is based on a hardened version of Windows and measures about 27-by-24-by-16 inches, costs about $245,000. RapidHIT boxes are already in use with intelligence agencies, says Jovanovich, who adds he's not at liberty to say which ones or what they're doing with them.

The FBI, which is believed to have upwards of 10 million DNA records on individuals already stored in databases, anticipates a significant expansion of DNA collection by means of Rapid DNA equipment.

The FBI has been known for pioneering a massive collection of fingerprint images and an online matching system that can be accessed remotely to help local law enforcement, as well as the Department of Defense and other law-enforcement agencies, nail down the identities of criminals and terrorists. Today, Dr. Alice Isenberg, chief of the biometrics analysis section at the FBI laboratory, explained in her presentation how the FBI hopes to expand the national DNA database used to investigate crime for DNA matches online as well.

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FBI eager to embrace mobile 'Rapid DNA' testing

18 September 2012 Last updated at 09:26 ET

A double rapist caught by chance years later through a DNA sample has been jailed for seven-and-a-half years.

Peter Hendry, 41, from Springburn in Glasgow, attacked the two women, who were working as prostitutes, in Rutherglen in 2004 and 2007.

He avoided capture until a former partner accused him of domestic abuse in 2011. DNA samples were taken which matched with both attacks.

Hendry, who denied the charges, was found guilty on a unanimous verdict.

Solicitor advocate Murray Macara QC, who represented the fork-lift truck driver, told the court that his client had "constantly and consistently maintained that he did not rape these two women".

Both women had told how Hendry picked them up and drove them to a deserted industrial estate.

A 33-year-old woman told the trial that he took her there in September 2004, pinned her down and raped her, before leaving her "like a bit of trash".

The woman reported the incident to police, but Hendry remained at large.

He carried out a similar attack on a 29-year-old woman in July 2007.

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Rapist caught by DNA test jailed