Gene cloning

Các kỹ thuật chính dùng trong công nghệ DNA tái tổ hợp 1.1. Khái niệm 1.2. Các enzym dùng trong công nghệ DNA tái tổ hợp 1.3. Các vector nhân dòng dùng trong công nghệ DNA tái tổ hợp 1.4. Nhân dòng gen (gene cloning) 1.5. Chọn lọc, tạo dòng và sự biểu hiện của gen 2. Các kỹ thuật chính sử dụng trong phân tích DNA 2.1. Kỹ thuật chiết tách DNA và RNA 2.2. Kỹ thuật tạo ngân hàng cDNA 2.3. Phương pháp PCR 2.4. Kỹ thuật xác định trình tự DNA 2.5. Kỹ thuật RFLP (dựa vào lai DNA/DNA) 2.6....

46p hoatuoi0939310 26-02-2013 283 81   Download

Bài giảng "Công nghệ di truyền - Chương 3: Simple cloning" cung cấp cho người học các kiến thức: Vector pUC, vai trò của các gene, cắt vector và DNA, nối bằng ligase được, chuyển tất cả cấu trúc vào E.coli, Operon lacZ và IPTG, chọn dòng mang DNA tái tổ hợp,... Mời các bạn cùng tham khảo nội dung chi tiết.

55p doinhugiobay_10 12-01-2016 111 19   Download

Bài giảng "Simple cloning" cung cấp cho người học các kiến thức: Vector pUC, vai trò của các gene, cấu trúc của các gen, cắt vector và DNA, nối bằng ligase được, chuyển tất cả cấu trúc vào E.coli, chọn dòng mang DNA tái tổ hợp, vector nhân tạo đầu tiên, tế bào chủ, tính chất của tế bào chủ, nhân dòng sản phẩm PCR,... Mời các bạn cùng tham khảo.

55p doinhugiobay_12 15-01-2016 56 5   Download

An optogenetic upgrade for the tet - OFF system fused the inhibitor to the blue light-responsive B-LID degron and optimized the performance of this construct with regard to the number of Med25VBD repeats. This approach resulted in an optogenetic upgrade of the popular Tet-OFF (TetR-VP64, tetO7-PhCMVmin)  system  that  allows tunable, blue light-inducible gene expression in HEK-293T  cells.

5p thuyancn 01-06-2015 30 5   Download

The plastid transcription kinase (PTK), a component of the major RNA polymerase complex from mustard chloroplasts, has been implicated in redox-mediated regulation of plastid gene expression. A cloning strategy to define the PTK gene(s) resulted in the isolation of a full-length cDNA for a protein with overall high homology with the a subunit of cytosolic casein kinase (CK2) that contained an N-terminal extension for a putative plastid transit peptide.

9p system191 01-06-2013 46 4   Download

We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124–126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of  52 kb. The first intron is  16 kb in length and is followed by an unusually large exon ( 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences....

10p system191 01-06-2013 46 4   Download

Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.

8p system191 01-06-2013 36 4   Download

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two-hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

10p research12 01-06-2013 40 3   Download

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best-characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. ...

7p research12 01-06-2013 35 3   Download

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide.

10p research12 01-06-2013 48 4   Download

We report the cloning and functional charaterization of the full-length cDNA and gene encoding a Toxoplasma gondii DNA repair enzyme designated TgDRE. The gene is composed of three exons separated by two introns of 780 and 630 bp, and encodes a protein with a predicted molecular mass of 49.6 kDa. The native TgDRE protein, with a molecular mass of 60 kDa, is only detected in the virulent tachyzoite stage of T. gondii. However, the transcript is present in both asexual parasite stages, virulent tachyzoite and avirulent encysted bradyzoite. ...

9p research12 01-06-2013 35 5   Download

Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP-A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse.

10p research12 01-06-2013 42 6   Download

A gene having high sequence homology (45–49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer....

8p research12 01-06-2013 27 4   Download

Sugar conjugation is a major pathway for the inactivation and excretion of both endogenous and exogenous compounds. We report here the molecular cloning and functional characterization of a phenol UDP-glucosyltransferase (UGT) from the silkworm, Bombyx mori, which was named BmUGT1. The complete cDNA clone is 1.6 kb, and the gene is expressed in several tissues of fifth-instar larvae, including fat body, midgut, integument, testis, silk gland and haemocytes. The predicted protein comprises 520 amino acids and has  30% overall amino-acid identity with other members of the UGT family.

7p research12 01-06-2013 39 4   Download

We found a leucine aminopeptidase (LAP; EC 3.4.11.1) to be abundant in meiotic prophase tissue of a basidiomycete, Coprinus cinereus. After direct puri®cation of the amino-peptidase component from meiocytes, we cloned the gene by degenerate PCR using partial amino-acid sequences of the puri®ed enzyme and 5¢ and 3¢ RACE. It was homologous to the eukaryotic leucine aminopeptidase gene.

7p research12 01-06-2013 46 6   Download

AnN-acyl-D-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-D-methionine. The bac-terium was classified as Variovorax paradoxusby phylo-genetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bpORF encoding a polypeptide of 488 amino acids. TheV.

11p research12 29-04-2013 44 3   Download

TheatpBandatpF genes ofPropionigenium modestumwere cloned as His-tag fusion constructs and expressed in Escherichia coli.Both recombinant subunits a and b were purified via Ni 2+ chelate affinitychromatography. A func-tionallyactive Focomplex was reassembledin vitro from subunits a, band c, and incorporated into liposomes.

7p research12 29-04-2013 28 3   Download

Osmoregulated periplasmic glucans (OPGs) ofRhodobacter sphaeroidesare anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is pro-vided by the substitution of the glucosidic backbone by succinyl residues. A wild-type strain was subject to trans-poson mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatogra-phy.

12p research12 29-04-2013 43 6   Download

Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning functionof the hsp70s.We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNAproduced by the same gene in that it contains a long 3¢ untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation.

8p research12 29-04-2013 34 3   Download

We have cloned the gene coding for theBacillus subtilis glycine oxidase (GO), a new ¯avoprotein that oxidizes gly-cine and sarcosine to the corresponding a-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expressionvectorpT7.7weproducedarecombinantplasmid (pT7-GO).ThepT7-GOencodes a fullyactive fusionprotein with six additional residues at the N-terminus of GO (MARIRA).

8p research12 29-04-2013 38 3   Download