* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Recombinant DNA and Biotechnology
DNA methylation wikipedia , lookup
DNA sequencing wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
DNA barcoding wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Epigenetics wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA profiling wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genetic engineering wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA polymerase wikipedia , lookup
Designer baby wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Point mutation wikipedia , lookup
Genealogical DNA test wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Microevolution wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genome editing wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Non-coding DNA wikipedia , lookup
Epigenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Primary transcript wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
DNA supercoil wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genomic library wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
DNA vaccination wikipedia , lookup
Helitron (biology) wikipedia , lookup
Molecular cloning wikipedia , lookup
Recombinant DNA and Biotechnology Nucleicacidfunction:CentralDogma 1 The structure of the information: 5' Flanking GENE: prokaryotes eukaryotes +1 –35 Teminator Coding Region Promoter Exon 1 –10 Intron 1 3'-flanking Exon 2 Other elements TATA box Start of Transcription mRNA: prokaryotes eukaryotes AUG 5’-UTR Shine-Delgarno End of Transcription Example: Stop Codon OPEN READING FRAME (ORF) 3’-UTR 3’-Poly(A) 5’-cap PROTEIN: prokaryotes Spliced Start of Translation N-Term End of Translation C-Term eukaryotes Nucleicacidfunction:CentralDogma Exceptions to the direction of the information flow: • RNA editing – change in ORF sequence, but not from DNA • Reverse transcription – making DNA from RNA (retroviruses) 2 • RNA editing – change in ORF not from DNA • Reverse transcription – making DNA from RNA (retroviruses) Used to make copy DNA (cDNA), which is a copy of the mRNA 3 Control of Gene Expression: Eukaryotes Eukaryotic gene regulation can occur at multiple points in transcription and translation: •Initiation of transcription wActivation of transcriptional initiation wChromatin remodeling •mRNA processing •mRNA transport •mRNA stability •Initiation of translation •Post-translational controls •Protein stability prokaryotes eukaryotes Recombinant DNA and Biotechnology Recombinant DNA is DNA made in the laboratory that is derived from at least two genetic sources. Recombinant DNA has allowed molecular biology to come full circle. FUNCTION Biochemistry Genetics PROTEIN GENE rDNA Recombinant DNA has one simple goal: MAKE MORE 4 Recombinant DNA and Biotechnology Recombinant Proteins Recombinant DNA and Biotechnology Recombinant DNA is DNA made in the laboratory that is derived from at least two genetic sources. • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants - Expression - Site-directed mutagenesis 5 Recombinant DNA and Biotechnology Restriction enzymes are used to cut DNA into fragments, which then are spliced together in new combinations. DNA ligase catalyzes the joining of DNA fragments. Recombinant DNA and Biotechnology Restriction Sites Restriction enzymes recognize palindromic DNA sequences: 5ʼ…….GAATTC……3ʼ 3ʼ…….CTTAAG……5ʼ Some make straight cuts, others make staggered cuts, resulting in overhangs or sticky ends. 6 Recombinant DNA and Biotechnology Restriction Endonucleases EcoRI Endonuclease EcoRI endonuclease PDBid 1ERI EcoRV Endonuclease EcoRV endonuclease PDBid 4RVE Restriction Endonucleases Cleave at Specific Recognition Sites 7 Restriction Endonucleases Key to Making rDNA Molecules DNALigasewill“seal”the“nick”by makingthecovalent phosphodiesterbond DNA Ligase: Also Key to Making rDNA Molecules 8 DNA Ligase Reaction Restriction Endonucleases & DNA Ligase: Key to Making rDNA Molecules Process Diagram: Recombinant DNA Construction Recombinant DNA molecule Where does this Foreign DNA come from and how is it purified? 9 Recombinant DNA and Biotechnology fragments used for molecular cloning come from two sources: Vectors and Inserts DNA • Genomic DNA • cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA Represents an mRNA from a given cell/tissue. cDNA is produced by making a DNA copy of the mRNA population using the RNAdirected DNA polymerase called, reverse transcriptase. A genomic clone contains the gene(s) as a fragment of the genome of an organism. The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector. A genomic clone A cDNA library is a “snapshot” of the transcription pattern of the cell. cDNA clones are used to provide the ORF for expressing the protein A cDNA clone Recombinant DNA and Biotechnology Vectors and Inserts: cDNA Cloning 10 Recombinant DNA and Biotechnology Vectors and Inserts: cDNA Cloning How do you find your DNA of interest? Restriction Digest Electrophoretogram Process Diagram: Southern Blotting Using polynucleotide kinase and g32P-ATP 11 Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants - Expression - Site-directed mutagenesis All vectors have 3 things: 1. Autonomous replication ability 2. Selection for hosts that contain vector 3. Site for insertion of rDNA Viruses as vectors (bacteriophage or retroviruses). • Viruses can be altered to attenuate some detrimental genes in the virus (e.g., those that kill the host). • Viruses can be altered to carry recombinant DNA into cells Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA Inserting the recombinant DNA into a cell: (rDNA) • Cells may be treated with chemicals to make plasma membranes more - Transformation of hosts permeable—DNA diffuses into cells. - Selection of transformants • Electroporation—a short electric shock Transformation: Recombinant DNA is cloned creates temporary pores in membranes, - Expression by inserting it into host cells (transfection if and DNA shoots to the + end and can enter cells. Site-directed mutagenesis host-cells are from an animal). Second key discovery in biotechnology. Usually only a few cells are transformed (1 cell in 10,000). Reason for the need for a selectable marker. The first host cells used were bacteria, especially E. coli. Yeasts (Saccharomyces) are commonly used as eukaryotic hosts. 12 Recombinant DNA and Biotechnology • Biochemical Basis of Biotechnology - Restriction enzymes, DNA ligase - Vectors and Inserts to make recombinant DNA (rDNA) - Transformation of hosts - Selection of transformants • Use ofExpression antibiotic resistance gene (e.g., ampicilin resistance) on a plasmid mutagenesis - Site-directed • For viral vectors, use of “infected” phenotype. • Use of “selectable markers” to detect either insertion into the vector or incorporation into the host. Some of these are a type of reporter gene—a gene whose expression is easily observed. • Many plasmids contain the lacZ gene with a multiple cloning site within its sequence. lacZ codes for an enzyme that can convert the substrate X-Gal into a bright blue product Plac Insert ORF of interest • Originofreplication • Antibioticresistancegene • Multiplecloningsite • Promoterfortranscription, translation(hostspecific) Recombinant DNA Clone Ori –Insert – Makes b-Galactosidase +Insert – Doesn’t make b-Galactosidase 13