generation alignment and variant calling

... formation of double strands and the library preparation procedure that precedes sequencing Protocols for the creation of double-stranded Page 14 of 18 oligonucleotide pools and preparation of libraries ... reaction composed of purified protein, doublestranded DNA ligand and 125 μg/μl poly dI-dC buffered in 12 mM HEPES pH7.8, 75 mM KCl, mM EDTA, mM GTP and 12.5% glycerol was incubated at room temperature ... experimental work and interpretation of data DS supplied material MLB and TS supplied auxiliary data and contributed to discussions IAU conceived the study, supplied material and contributed to...

Ngày tải lên: 09/08/2014, 23:20

... were sonicated and centrifuged and the bacterial pellet was dissolved in a buffer containing guanidine-HCl and reduced glutathione The sample was again sonicated and centrifuged, and the supernatant ... difluoride)-membrane and subjected to Western blot using MoAb 44 (A) Lane and show the Western blot, whereas lane and show the corresponding silver stained gels of two different C4BP preparations (lanes and 3, and ... flow rate used was 0.5 mL per minute and 0.5 mL fractions were collected Fractions were chosen and pooled based on analysis of silver staining after SDS/PAGE and stored at )70 °C until further...

Ngày tải lên: 17/03/2014, 09:20

... fluoride and 10% glycerol For helicase and ATPase assays, HpSSB (Wt) and DC20SSB were dialysed against MonoQ and MonoS buffers and subjected to ion exchange chromatography using MonoQ and MonoS ... Wt and DC20 SSB protein in varying concentrations (0, 0.45, 0.9, 1.8, 2.7 and 3.6 lg, respectively) with 300 ng of M13mp18 single-stranded circular DNA and ⁄ or 300 ng of pUC18 double-stranded ... HpSSB and biochemical properties (A) The amino acid sequences of E coli, B subtilis and H pylori were aligned using the CLUSTALW multiple sequence alignment programme *Identical residues; ‘:’ and...

Ngày tải lên: 30/03/2014, 02:20

... capping functions and that regulate telomere length [3] The TRF1, TRF2 and Pot1 subunits bind to telomeric DNA and to the other subunits of the complex, namely the TIN2, TPP1 and Rap1 proteins ... transformed T lymphocytes and in Tax-expressing lymphocytes, the transcriptional balance between hTERT and the shelterin subunits TRF1, TRF2 and Pot1 are regulating telomere homeostasis and cell proliferation ... the transcription of telomerase and that of POT1, TERF1 and TERF2 genes Transcriptional expression of hTERT, POT1, TERF1 and TERF2 genes in HTLV-1 transformed and Tax-expressing T lymphocytes...

Ngày tải lên: 13/08/2014, 09:21

Ngày tải lên: 17/07/2015, 20:31

... 1ị where SD and SRF are the respective areas under the emission spectra of the donor and a reference compound, and ARF and AD are the respective absorbances of the reference compound and donor ... 10PỵDAị=2 2ị where F and Fcor are uorescence intensity before and after the correction, and P and DA denote the initial sample absorbance at the excitation wavelength and the change in absorbance ... bold An equimolar amount of complementary strand was added, and the mixture was heated for at 96 C and slowly cooled to room temperature The double-stranded DNA was stored at )20 C in experimental...

Ngày tải lên: 20/02/2014, 23:20

... mutants and (each with 3· GC and five mutations) Both mutants and were bound equally well by HbpR under the conditions of EMSA Changing, more or less randomly, other sets of bp (mutants 10, 11 and ... (lanes 7–10) Lanes and 6, no CBP fusion protein added; lanes and 7, 200 nM CBP fusion protein; lanes and 8, 400 nM CBP fusion protein; lanes and 9, 800 nM CBP fusion protein; lanes and 10, 1200 nM ... sequence’ (UAS) and is characterized by two imperfect palindromes of 16 bp and a spacing of between 29 and 42 bp between their centres (Fig 1A) [20] The regions covered by XylR on the Pu and of HbpR...

Ngày tải lên: 07/03/2014, 17:20

... Kd (K rel ) and kcat (k rel ) values for binding and d cat methylation of DNA containing (+)-cis-B[a]PN2-dG and (+)-trans-B[a]P-N2-dG adducts by M.SssI (A) and M.HhaI (B) The K rel and k rel d ... and Moscow State University), NIH Grant CA 099194 (New York University), and RFBR Grants 04-04-49488 and 05-0449690 (Moscow State University) We thank Dr B Jack and Dr G Wilson from New England ... M.SssI and M.HhaI The rates of methylation of the X+CG ⁄ CGC, GCX+ ⁄ CGC, X+CG ⁄ CGM and GCX+ ⁄ CGM duplexes by SssI and HhaI MTases were determined under steadystate conditions (Fig 4), and the...

Ngày tải lên: 19/02/2014, 00:20

... one strand and two AG steps in the other The mdm2 target, whose interaction with p53 was most strongly affected by DNA cis-platination, contains GG, GGG and AG motifs in one strand and GG and GTG ... with hemagglutinin (HA), we used antibody to HA and obtained analogous band patterns to those obtained with p53 (Fig 2; lanes 6–7, bands SR73b and SR73d), confirming the specificity of the observed ... sites (p53DBSs) mdm2 and p21, and the synthetic PGM1 sequence; (B) mutated p53DBSs PGM4, p21a and p21b (Fig 1) The top panels show sections of autoradiograms showing the R53 bands corresponding...

Ngày tải lên: 19/02/2014, 05:20

... (PfRad51), and ScRad51 around the L1 and L2 loops The L1 and L2 loops, which are invisible in the crystal structure of the ATPase domain of HsRad51 [21], are represented by boxes, and the Y232 and F279 ... 21, and 24), and lM (lanes 4, 7, 10, 13, 16, 19, 22, and 25) Lane indicates a negative control experiment without the Rad51 protein Joint molecules and nicked circular DNA are indicated by jm and ... 2-mercaptoethanol, and 10% (v ⁄ v) glycerol, and the flow rate was 0.5 mLÆmin)1 CD measurements DNAs The /X174 phage ssDNA and dsDNA used in the DNA binding and ATPase assays were purchased from New England...

Ngày tải lên: 19/02/2014, 06:20

... strand) and )33G, )35G, )46G, )56G and )68G (top strand) (Fig 3B) All the protected guanine bases except )56G are located in and around O1 and O2 Interestingly, )35G, )41G and )43G in O1 and ... to )48 and )52 to )87 regions of the top strand and )24 to )53 and )58 to )87 regions of the bottom strand were protected by CI (Fig 3B) The centers of these two sites harbor the 15 bp O1 and O2, ... QIAquick Gel Extraction Kit (Qiagen) The 34 bp O1, and 49 bp O2 DNAs (Fig 2A) were prepared by mixing and annealing primers PCR11 and PCR21 and IIa and IIb, respectively (Table 1) The cooperativity...

Ngày tải lên: 07/03/2014, 00:20

... (to delete site I), C and D (to delete site II), E and F (to delete site III), G and H (to delete the perfect inverted repeat) and I and F (to delete simultaneously sites II and III) were ligated ... Germany) and XL1-Blue [38], and the plasmids pET21a (Novagen) and pASK-IBA7 (IBA, Gottingen, ¨ Germany), were used for routine cloning purposes The constructs pUKS10 (pUC18 derivative) and pWKB1 ... with m Tris ⁄ HCl (pH 7.5), and the third with m HCl Subsequently, the gels were dried and exposed to an X-ray film DNA and oligonucleotide labeling PCR fragments and oligonucleotides were labeled...

Ngày tải lên: 07/03/2014, 10:20

... and Ser114) mutated in dHAND-Ala-X Akt binds to dHAND in vitro and in vivo To examine whether Akt can directly interact with dHAND, we performed pull-down assay using GST- Ó FEBS 2004 dHAND and ... mutants dHAND-Ala-X and dHAND-AlaXZ (Fig 4B) These results indicated that the major phosphorylation sites of dHAND were located within Fig Analysis of the interaction between dHAND and Akt (A) ... Coimmunoprecipitation assay of dHAND and Akt AU5-dHAND was immunoprecipitated from the cell lysate of 293T cells expressing AU5-dHAND and HA-Akt These samples were applied on 10% SDS/PAGE and bound proteins...

Ngày tải lên: 07/03/2014, 16:20

... sequence boxes I-III (CSB), tRNA genes, L and H strand promoters (LSP and HSP), L and H strand transcription start sites (LSS and HSS) The L-strand sequence and position of the putative promoter ... OL and OH represent the origin of L and H strand replication, respectively LSS, L strand start site; HSS, H strand start site The map positions of the rRNA, tRNAs and mRNAs (ND1 to ND6, A6 and ... electro eluted and digested with DNAse1 as described in Materials and methods Maxam and Gilbert sequencing reactions of the D-TERM probe (GA and CT) were used as markers in lanes and A vertical...

Ngày tải lên: 08/03/2014, 08:20

... tube containing two layers of caesium chloride (5% and 40%), and ultracentrifuged for 16 h at 36 000 g at 15 °C The virus band was collected and DCV was recovered by centrifugation (25 000 g, ... kept on standard medium at 22 °C, and counted daily Microscopic observations For observation of GFP expression patterns, live flies and larvae were anaesthetized with ether or on ice, and viewed ... fluorescence in noninfected larvae in both anterior and posterior spiracles, in the digestive tract, the ring gland, the antenna buds and testis (Fig 3A,B and data not shown) In adult flies, GFP was expressed...

Ngày tải lên: 08/03/2014, 08:20

... Fig Isolation and identification of LBP L donovani membrane proteins isolated by biotinylation and streptavidin–agarose extraction and passed through DEAE-cellulose, Con A–Sepharose and laminin– ... donovani using anti-LBP Ig and protein A–Sepharose beads When these immune complexes were dissociated and run on SDS/PAGE and autoradiographed, we observed a single band at 67 kDa (lane 6) RESULTS ... different pH levels: pH 6.5 and 7.0 (20 mM phosphate), pH 7.5 and 8.0 (20 mM Tris/HCl) and pH 9.0 (20 mM glycine/NaOH) with usual amount of NaCl (150 mM) Dissociation constants and binding maxima (where...

Ngày tải lên: 08/03/2014, 23:20

... BcBL2 and BcBL3, and the effects of nucleotides on RNA binding A rapid and convenient filter binding assay [14] was used for many of these experiments Electrophoretic mobility shift assays and sedimentation ... and PRPP) or inhibition (GMP and GTP) of binding of PyrR to BcBL1 and BcBL2 Data are the average of at least two independent determinations To learn more about how PyrR distinguishes purine and ... was determined from both sedimentation velocity and equilibrium sedimentation experiments at high and low protein concentrations and in the presence and absence of 0.1 m NaCl The results of the...

Ngày tải lên: 16/03/2014, 06:20

... mobility controls: labelled Comp-CT alone and a completely annealed reaction Samples were taken at 0, 1, 3, 10, and 30 Single-stranded (ss) and double-stranded (ds) species are indicated (B) Bar ... 1· Gels were dried and autoradiography visualized by using GP-Storage Phosphor Screen and storm scanner and software (Amersham Biosciences) Radioactive bands were quantified and annealing percentage ... test) protein that binds single-stranded nucleic acids and is able to promote interstrand reannealing of DNA as well as RNA [35,36] hnRNP Al is phosphorylated by CK2 and PKA PKA phosphorylation suppresses...

Ngày tải lên: 16/03/2014, 12:20

... was incubated in binding buffer in the absence (lanes and 3) or presence of 75 lM ssDNA (lanes and 5) and mM ATP (lanes and 5) for h at 37 °C and then subjected to partial digestion with trypsin ... Disassembly and recruitment of hRad51 and hRad52 proteins shorter and thicker nucleoprotein filaments when ssDNA is added [28] Tumour suppressors BRCA2 and p53 interact with the ... in lanes and with those in lanes 3, and reveals that the FEBS Journal 273 (2006) 199–207 ª 2005 The Authors Journal compilation ª 2005 FEBS 203 Disassembly and recruitment of hRad51 and hRad52...

Ngày tải lên: 16/03/2014, 14:20

... the bands corresponding to single strands of DNA and interstrand cross-linked duplex were quantified Rearrangement of intrastrand cross-links The platinated oligodeoxyribonucleotide (top strand ... gel; concentrations of the oligonucleotide duplex and 2513-basepair fragment were 1.6 and 10 lgÆmL)1 (1.26 · 10)7 and · 10)9 M), respectively, and concentration of p53 was 3.9 · 10)8 M rb values: ... revealed that the major bands resulting from termination of RNA synthesis by the adducts of transplatin and trans-PtHMP were similar, appeared mainly at G and C sites and in a considerably less...

Ngày tải lên: 16/03/2014, 14:20