Scientists have obtained the first detailed molecular structure of a member of the Tet family of enzymes.
The finding is important for the field of epigenetics because Tet enzymes chemically modify DNA, changing signposts that tell the cell's machinery "this gene is shut off" into other signs that say "ready for a change."
Tet enzymes' roles have come to light only in the last five years; they are needed for stem cells to maintain their multipotent state, and are involved in early embryonic and brain development and in cancer.
The results, which could help scientists understand how Tet enzymes are regulated and look for drugs that manipulate them, are scheduled for publication in Nature.
Researchers led by Xiaodong Cheng, PhD, determined the structure of a Tet family member from Naegleria gruberi by X-ray crystallography. The structure shows how the enzyme interacts with its target DNA, bending the double helix and flipping out the base that is to be modified.

This is the structure of the Tet enzyme with DNA. Note the purple ball at the active site, close to which one DNA base is flipped out of the double helix. Also note the degree to which the double helix is bent. Credit: Xiaodong Cheng, Emory University

"This base flipping mechanism is also used by other enzymes that modify and repair DNA, but we can see from the structure that the Tet family enzymes interact with the DNA in a distinct way," Cheng says.
Cheng is professor of biochemistry at Emory University School of Medicine and a Georgia Research Alliance Eminent Scholar. The first author of the paper is research associate Hideharu Hashimoto, PhD. A team led by Yu Zheng, PhD, a senior research scientist at New England Biolabs, contributed to the paper by analyzing the enzymatic activity of Tet using liquid chromatography–mass spectrometry.
Using oxygen, Tet enzymes change 5-methylcytosine into 5-hydroxymethylcytosine and other oxidized forms of methylcytosine. 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) are both epigenetic modifications of DNA, which change how DNA is regulated without altering the letters of the genetic code itself.
5-mC is generally found on genes that are turned off or on repetitive regions of the genome. 5-mC helps shut off genes that aren't supposed to be turned on (depending on the cell type) and changes in 5-mC's distribution underpin a healthy cell's transformation into a cancer cell.
In contrast to 5-mC, 5-hmC appears to be enriched on active genes, especially in brain cells. Having a Tet enzyme form 5-hmC seems to be a way for cells to erase or at least modify the "off" signal provided by 5-mC, although the functions of 5-hmC are an active topic of investigation, Cheng says.
Alterations of the Tet enzymes have been found in forms of leukemia, so having information on the enzymes' molecular structure could help scientists design drugs that interfere with them.
N. gruberi is a single-celled organism found in soil or fresh water that can take the form of an amoeba or a flagellate; its close relative N. fowleri can cause deadly brain infections. Cheng says his team chose to study the enzyme from Naegleria because it was smaller and simpler and thus easier to crystallize than mammalian forms of the enzyme, yet still resembles mammalian forms in protein sequence.
Mammalian Tet enzymes appear to have an additional regulatory domain that the Naegleria forms do not; understanding how that domain works will be a new puzzle opened up by having the Naegleria structure, Cheng says.

At Tokuda Hospital in Sofia was presented the latest genetic testing in prenatal diagnosis.
 
Latest biomedical innovation in prenatal care for expectant mothers called Prenatest. This is the first non-invasive prenatal test in Bulgaria. Unlike other Prenatal tests Prenatest analyze the DNA of the fetus without the manipulation of the body of the mother and fetus.

The study gives as early safest information about the most common forms of chromosomal abnormalities in the fetus: trisomy 21, 18 and 13 - Down syndrome, Edwards syndrome and Patau syndrome. Subjecting Prenatest can be done by the 9th week of gestation and accuracy of the results is 99%.

Europe territory test is licensed by the German company Layfkodeks. He carries CE mark, making it the only certified genetic testing of third generation - non-invasive prenatal test within the European Union.
 
Breaking study was developed based on the discovery that the blood of a pregnant woman circulating DNA of the baby. This DNA is extracellular or free. Test its patented technology that isolated from maternal blood free fetal DNA and makes it possible for her research. Blood samples were transported to the laboratory Layfkodeks in Germany, where it is subjected to high-tech genetic analysis.

If the gene has a similar role in humans will be able to develop new screening tests

British scientists have identified a gene in mice, which increases the risk of ovarian cancer, as defective.

Rodents lacking gene are twice as likely to develop cancer, ovarian cancer, and to show signs of infertility. If the gene has a similar role in humans will be able to develop new screening tests, said scientists from the charity Cancer Research UK.

They focus on gene Helq, who was involved in the restoration of damaged DNA. It turned out that mice deprived of it, are two-fold higher risk of ovarian cancer.

"The results show that if there are problems with Helq in mice increases the likelihood of developing ovarian cancer and other tumors, said study leader Dr. Simon Boultan. This is exciting because it is possible the same effect was observed in women with a defective gene Helq. The next step is to check whether this is so."

AccuPower® ProFi Taq PCR PreMix for high efficiency and amplification of long range PCR.

AccuPower® ProFi Taq PCR PreMix is a convenient lyophilized PCR master mix containing ProFi Taq DNA polymerase, reaction buffer, dNTPs, tracking dye, and a patented stabilizer. ProFi Taq DNA polymerase in the premix is a unique recombinant Taq DNA polymerase that offers enhanced amplification efficiency and higher fidelity for PCR. AccuPower® ProFi Taq PCR PreMix is applicable to any template DNA, and especially effective in amplifying large genomic DNA fragments around 20 kb. AccuPower® ProFi Taq PCR PreMix provides accurate long-range amplification of standard and amplification of low-copy target, and is highly suitable for all PCR applications.

Features and Benefits

Specifications

5' to 3' exonuclease: Yes
3' to 5' exonuclease: Yes
3' – A Overhang: Yes
PCR product size: ~ 30kb

Application

- Primer extension
- long-range amplification from genomic DNA
- High amplification efficiency
- Excellent performance on difficult templates
- Amplification of low-copy targets
- High yield and high sensitivity PCR

Experimental data

Figure 1. Comparison of PCR amplification efficiency between AccuPower® ProFi Taq PCR PreMix from Gentaur and other suppliers' PCR master mix

The cycling conditions for AccuPower® ProFi Taq PCR PreMix were 95°C for 5min, 30 cycles of 95°C for 20 sec, 55°C for 20 sec and 72°C for 30 sec. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Target : human insulin receptor gene.
Lane M : 100 bp DNA Ladder(Gentaur, Cat. No. D-1030)
Lane 1 : 10 ng of human genomic DNA
Lane 2 : 1 ng of human genomic DNA
Lane 3 : 100 pg of human genomic DNA
Lane 4 : 10 pg of human genomic DNA

Figure 2. Comparison of PCR amplification efficiency between AccuPower® ProFi Taq PCR PreMix from Gentaur and other suppliers' PCR master mix

cDNA synthesized from 10-fold serial-diluted human total RNA from 10 ng to 10 pg using AccuPower® RocketScript™ Cycle RT PreMix. (Gentaur, Cat. No K-2201) wase used as a template for PCR amplification. The cycling conditions for AccuPower® ProFi Taq PCR PreMix were 95°C for 5min, 33 cycles of 95°C for 20 sec, 55°C for 20 sec and 72°C for 30 sec. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Target : human GAPDH gene.
Lane M : 100 bp DNA Ladder(Gentaur, Cat. No. D-1030)
Lane 1 : 10 ng of human total cDNA
Lane 2 : 1 ng of human total cDNA
Lane 3 : 100 pg of human total cDNA
Lane 4 : 10 pg of human total cDNA

Figure 3. Comparison of PCR amplification of long targets between AccuPower® ProFi Taq PCR PreMix from Gentaur and other suppliers' PCR master mix

The cycling conditions for AccuPower® ProFi Taq PCR PreMix were 95°C for 5 min, 30 cycles of 95°C for 20 sec, 65°C for 20 sec and 68°C for 4 min. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Lane M1 : Lambda/Hind III marker (Gentaur, Cat. No. D-1050)
Lane M2 : 1 kb DNA Ladder (Gentaur, Cat. No. D-1040)
Lane 1 : 2 kb fragment (human tumor protein p53 gene)
Lane 2 : 3 kb fragment (human tumor protein p53 gene)
Lane 3 : 4.5kb fragment (human DNA cross-link repair 1A gene)
Lane 4 : 8 kb fragment (human hemoglobin epsilon 1 gene)

Figure 4. Comparison of PCR amplification of long targets between AccuPower® ProFi Taq PCR PreMix from Gentaur and other suppliers' PCR master mix

The cycling conditions for AccuPower® ProFi Taq PCR PreMix were 95°C for 5 min, 32 cycles of 95°C for 20 sec and 68°C for 15 min. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol. Human genomic DNA was used as a template for PCR amplification.
Lane M1 : Lambda/Hind III marker (Gentaur, Cat. No. D-1050)
Lane M2 : 1 kb DNA Ladder (Gentaur, Cat. No. D-1040)
Lane 1 : 11 kb fragment
Lane 2 : 13.5 kb fragment
Lane 3 : 17.6 kb fragment
Lane 4 : 21.4 kb fragment

Figure 5. Comparison of PCR amplification of long targets between AccuPower® ProFi Taq PCR PreMix from Gentaur and other suppliers' PCR master mix

The cycling conditions for AccuPower® ProFi Taq PCR PreMix were 95°C for 5 min, 32 cycles of 95°C for 20 sec, 65°C for40 sec, and 68°C for 20 min. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol. Lambda DNA was used as a template for PCR amplification.
Lane M1 : Lambda/Hind III marker (Gentaur, Cat. No. D-1050)
Lane M2 : 1 kb DNA Ladder (Gentaur, Cat. No. D-1040)
Lane 1 : 15 kb fragment
Lane 2 : 20 kb fragment
Lane 3 : 25 kb fragment
Lane 4 : 30 kb fragment

Applications

 

• Routine PCR applications
• TA cloning

Description

BIOTAQ™ is widely used by molecular biologists that have come to depend upon the robust performance of this reagent.

BIOTAQ is a highly purified thermostable DNA polymerase offering very high yield over a wide range of PCR templates (Fig. 1), and is the ideal choice for most assays. BIOTAQ is a robust preparation and consistently delivers high yields with minimal background. BIOTAQ possesses 5´-3´ exonuclease activity and leaves an ´A´ overhang such that the PCR product is suitable for effective integration into TA cloning vectors.

BIOTAQ is supplied with 10x NH₄-based Reaction Buffer, which provides optimal conditions for most experiments. Additional MgCl₂ is provided to allow reaction conditions to be adjusted to suit the template.

The specificity and performance of BIOTAQ can be further improved with the use of 2x PolyMate Additive which is designed for GC- or AT-rich DNA, "dirty" templates or sequences with a high level of secondary structure.

BIOTAQ™ DNA Polymerase is purified from Thermus aquaticus.
 

Unit Definition

One unit will incorporate 10nmoles of dNTPs in 30min at 72°C.

Concentration

5u/µl

Storage Conditions

BIOTAQ can be stored for 12 months at -20°C.
 

Product Citations

1. Amaral, I.P. & Johnston, I.A. J. Exp. Biol. 214, 2125-2139 (2011).
2. Coutinho, C.P., et al. Infect. Immun. 79, 2950-2960 (2011).
3. Lora, J., et al. PNAS 108, 5461-5465 (2011).
4. Palazón, A., et al. Can. Res. 71, 801-811 (2011).
5. del Hoyo, A. & Pedrola-Monfort, Plant Systemat. Evol. 273(3-4), 151-67 (2008).

Best price on the market: 118 Euro for 500 Units

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