Daily Archives: December 24, 2013

DNA replication is the process of producing two identical copies from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is composed of two strands and each strand of the original DNA molecule serves as template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.[3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bidirectionally from the origin. A number of proteins are associated with the replication fork which assist in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new DNA by adding complementary nucleotides to the template strand.

DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine, cytosine, guanine, and thymine, commonly abbreviated as A,C, G and T. Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nucleobases pointing inward. Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (stronger: three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single strand are called the "3' (three-prime) end" and the "5' (five-prime) end". By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is 5' end, while the right end of the sequence is the 3' end. The strands of the double helix are anti-parallel with one being 5' to 3', and the opposite strand 3' to 5'. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end of a DNA strand.

The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand.[4]

DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[6] DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. (Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates.) When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.[Note 1]

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added.[7] In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.[7]

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli.[8] During the period of exponential DNA increase at 37C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.[9] Thus DNA replication is both impressively fast and accurate.

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DNA replication - Wikipedia, the free encyclopedia

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--

Fluidigm Corporation (FLDM) today announced a universal sample prep workflow for single-cell DNA sequencing that runs on its C1TM Single-Cell Auto Prep System. This workflow streamlines targeted, whole exome and whole genome sequencing in heterogeneous cell populations and enables researchers to discover and screen somatic mutations, such as SNP, small indels, and translocations.

Somatic mutations are non-inherited, random mutations that are accumulated over time and may play an important role in the origin and progression of complex diseases, such as aging, cancer, immunity, and neurodegenerative disorders.

Somatic mutations are often masked in sequencing of bulk tissue, leaving researchers with the risk of missing important, causal variants that elucidate disease mechanisms. Understanding somatic mutations can help identify more effective therapies, said Gajus Worthington, Fluidigm president and chief executive officer. The C1 DNA Sequencing workflow is the first to fully automate cell handling, imaging, staining, and whole genome amplification, all at a single-cell level. It enables researchers with a comprehensive suite of single-cell sequencing applications they can use to identify and screen novel DNA variants from heterogeneous samples at unprecedented resolution and speed, he added.

Human leukemia, such as Acute Myeloid Leukemia (AML), is a genetically heterogeneous disease caused by the accumulation of somatic mutations in hematopoietic stem/progenitor cells. These mutations change the normal mechanisms of self-renewal, proliferation, and differentiation of cells in the blood and are highly variable between AML patients, said Paresh Vyas, MD/PhD and Hematologist at the MRC Molecular Hematology Unit, University of Oxford and Oxford Biomedical Research Centre. We can use the C1 DNA Sequencing workflow to detect genetic changes that identify clonal structures to more accurately classify tumors. This will lead to better understanding of prognosis including risk of recurrence and possibly even overall survival, Vyas explained.

From discovery of disease factors to validating the most effective treatment, researchers can now use the C1 Single-Cell DNA Sequencing workflow for:

The new workflow consists of the C1 Integrated Fluidic Circuits, C1 Reagent kit, and validated scripts, and also leverages the GE illustra GenomiPhi V2 DNA Amplification Kit for whole genome amplification. The C1 DNA Sequencing workflow will be further enhanced by Fluidigms SINGuLAR TM Analysis Toolset 3.0, which will include new features to filter, visualize, and rapidly identify biologically relevant variants. The toolset can also be used to create custom variant groups to fit the specific needs of any clinical researcher.

This workflow allows researchers to:

The targeted sequencing workflow is currently available for early access customers. The whole genome and whole exome applications is expected to be released in early 2014.

Use of Forward-Looking Statements

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Fluidigm Launches Single-Cell DNA Sequencing Workflow to Study Somatic Mutations in Heterogenous Samples

By WILSON RING/Associated Press/December 24, 2013

MONTPELIER, Vt. (AP) A Vermont man who killed a Stowe woman and remained free for 14 years while the victims parents urged the Legislature to create the DNA database that was used to identify him as their daughters killer, died in prison Tuesday.

Howard Godfrey, 67, of Kirby died in the medical unit of the Southern State Correctional Facility in Springfield, said Vermont Corrections Commissioner Andy Pallito. He did not give a cause of death, but said Godfreys death of natural causes was expected.

Godfrey was convicted in 2008 of the sexual assault and killing of Patricia Scoville, 28, and was serving a sentence of life without parole when he died.

Scovilles body was found in a shallow grave at the Moss Glen Falls, a scenic spot outside Stowe village. She had ridden her bicycle there on Oct. 23, 1991. Her body was found several days later. She had been hit in the back of the head and sexually assaulted.

Scovilles death went unsolved for years while her parents, Ann and David Scoville, of Canadaiga, N.Y., lobbied the Vermont Legislature to create a DNA database of people convicted of certain crimes.

David Scoville, reached at his New York home Tuesday, said Godfreys death marks the end of another chapter since the death of his daughter.

We always say there is no such thing as closure other than having Patty back, but this is a closure of sorts, said Scoville, who along with his wife continue to speak in favor of DNA database proposals since the Vermont law led to their daughters killer.

In 2002 the Scovilles received the National Crime Victim Service Award for their efforts. And after Godfreys 2008 sentencing, the Scovilles were honored by state officials for their efforts to enact Vermonts DNA databank. The states DNA laboratory was named in their daughters memory.

Godfrey gave a DNA sample in 2000 after he was convicted of a non-fatal aggravated assault of a woman in 1996 in Morrisville, not far from where Scoville was killed. That sample was not entered into the national database that linked him to Patricia Scovilles killing until 2005. He was arrested several days later.

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Man in killing that led to Vermont DNA law dies

DNA has linked a convicted burglar to a brutal night-time rape at a South Beach lifeguard stand in May.

Newly released court records show Blitz Santana Monestine, 29, is the chief suspect in the previously unpublicized attack. A second man remains unidentified.

Miami Beach detectives served a warrant recently to obtain a second DNA sample from Monestine, who is in a downtown federal detention center awaiting sentencing in an unrelated federal gun case.

So far, Monestine has not been charged with sexual battery as investigators await the results of the second DNA test.

According to a search warrant, the attack happened on May 5, when a woman in her 20s had just left the Club Duce Bar, at 222 14th St. The time: 4:30 a.m.

The woman admitted she was intoxicated at the time. But she remembered that a group of men outside the bar began to insult and scream at her, according to the warrant.

One of the men began walking with the woman toward the beach. At 17th Street, the man led her up the lifeguard stand. Suddenly, a cohort appeared and the men pinned her to the floor and began to rape her, according to the warrant.

Please stop, please stop. Please let me go, the woman told detectives she screamed.

If you scream you are going to die, she said one of the men told her.

One of the attackers choked her. She injured her hand as she fought, according to the search warrant filed in Miami-Dade circuit court by Miami Beach Detective Gustavo Sanchez.

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Suspect identified in brutal South Beach lifeguard-stand rape