Biology technology DNA recombinant technology

Molecular biotechnologies or Genetechonology really refers to: Molecular biology: is the study of biology at a molecular level Molecular genetics: is the field of biology and genetics that studies the structure and function of genes at a molecular level Genetic engineering: is the direct manipulation of an organism's genome using modern DNA technology Molecular Biology: DNA Recombinant DNA recombinant technology used in the isolation or synthesis and joining together of pieces of DNA These recombinant DNA molecules can then be introduced into bacteria, yeasts, or other cells where they can replicate and function (code for protein or functional RNAs) TOOL: Enzymes Host Cells Vectors The Application of DNA Recombinant Technology Express protein and study protein structure and its function in vivo; isolate and purify protein to study in vitro Use purified protein to make antibodies for medical purpose and/or make vaccines for the treatment of disease Scale-up production, isolation, and purification of therapeutic proteins (insulin, …) for use in humans as recombinant DNA products Study gene structure, gene sequence, and gene expression in organs, tissues and The Application of DNA Recombinant Technology Find chromosomal location of cloned gene, determine gene copy number, and study gene structure Use in human gene therapy, diagnose human genetic disorders and infectious disease conditions Create transgenic animals and gene knockout animals to study gene function Create new genetically engineered microorganisms, animals and plants with a range of applications from waste-degrading microorganisms to disease resistant plants Site-specific recombinases Site-specific recombinases cleave at specific sequences and rejoin DNA using a covalent protein-DNA intermediate   Tyrosine Recombinases Serine Recombinases Site-specific recombinases Serine Recombinases: introduce double-stranded breaks in DNA and then swap strands to promote recombination Tyrosine Recombinases: break and rejoin one pair of DNA strands at a time Serine Recombinases • First , the serine recombinases cleave all four strands • Second, DNA swap occurs • Finally, the serine recombinases are liberated and they seal the DNA strands Tyrosine Recombinases • Cleave and rejoin two DNA strands first, and then • Cleave and rejoin the other two strands Recombinase technology: applications Site-Specific Recombinases Molecular function of the type III effectors from Xanthomonas Molecular function of the type III effectors from Xanthomonas AvrBs3 AvrBs3 from Xanthomonas: Transcription Activator-Like (TAL) effectors Specific inducibility of the Hax boxes by Hax effectors GUS reporter constructs were codelivered via A.tumefaciens into N.benthamiana repeat-variable di-residue, RVD Overview of the CRISPR - Cas system Diversity of CRISPR - Cas systems CRISPR spacer acquisition Selection of the protospacer adjacent motif (PAM) Nicking of the leader-end repeat in the CRISPR locus Integration of the new spacer and duplication of the flanking repeat Biogenesis of crRNAs Type I, III Type II Primary processing of the pre-crRNA is catalysed by Cas6, which typically results in a crRNA with a 5ʹ handle of nucleotides, a central spacer sequence and a longer 3ʹ handle Secondary processing of the 3ʹ end of crRNA (yellow triangles) is catalysed by unknown ribonucleases In type II CRISPR–Cas systems, the repeat sequences of the pre-crRNA hybridize with complementary sequences of transactivating CRISPR RNA (tracrRNA) The double-stranded RNA is cleaved by RNase III (red triangles) and further trimming of the 5ʹ end of the spacer is carried out by additional nucleases (yellow triangle) Current Opinion in Biotechnology 2014, 29:46–54 Plot highlighting the increasing popularity of sRNAs, TALEs and CRISPR/Cas in the published primary literature over the last 13 years The system-CRISPR interference (CRISPRi): Interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding, Can be used to repress multiple target genes simultaneously, and its effects are reversible The CRISPR Interference System ...Molecular Biology: DNA Recombinant DNA recombinant technology used in the isolation or synthesis and joining together of pieces of DNA These recombinant DNA molecules can then be... (insulin, …) for use in humans as recombinant DNA products Study gene structure, gene sequence, and gene expression in organs, tissues and The Application of DNA Recombinant Technology Find chromosomal... (code for protein or functional RNAs) TOOL: Enzymes Host Cells Vectors The Application of DNA Recombinant Technology Express protein and study protein structure and its function in vivo; isolate