Basics of Recombinant DNA Technology Biochemistry 302. March 5, 2004 Bob Kelm

Transcription

1 Basics of Recombinant DNA Technology Biochemistry 302 March 5, 2004 Bob Kelm

2 Applications of recombinant DNA technology Mapping and identifying genes (DNA cloning) Propagating genes (DNA subcloning) Modifying genes (DNA mutagenesis) Expressing gene products (recombinant protein production) Reintroducing modified genes into organisms (transgenics)

3 Recombinant DNA:Experimental strategy Source DNA of interest Expression cloning Methods for isolating genes & sequences of interest hybridization techniques PCR library screening Specific gene or sequence Vector Recombinant DNA molecule Sequence analysis Functional studies of the gene (mutagenesis) Expression of gene products mutant gene Recombinant Protein Reintroduction into the original genome: transgenic technology

4 Propagating foreign DNA: Plasmids (autonomous, extrachromosomal circular dsdna molecules not required for growth but providing a selective advantage) Naturally occurring plasmids Typically large, low copy number, self-transmissible, and encoding genes that confer antibiotic resistance and/or unusual catabolism. Others are small and non-transmissible. Engineered plasmids (vectors) Small size, unique restriction sites (e.g. MCS) Selectable marker - drug resistance One or more ori (e.g. E. coli and yeast and phage) Screen for insertion

5 Engineered plasmids: cloning and expression vectors Fig. 25B.4

6 Type II restriction endonucleases (work horses of recombinant DNA technology) Table 25-2 Fig EcoRI Recognize simple symmetrical sequences Cut unmethylated DNA generating 3 OH & 5 -phosphate termini with either blunt or staggered ends Most are homodimers with subunits of kda and require a divalent cation for catalysis Counterpart methylase: recognizes un- and hemi-methylated DNA, AdoMet

7 Joining restriction fragments to create a recombinant DNA molecule Fig. 25B.1

8 A simple cloning experiment Digestion of vector and target DNA with same enzyme Purification of products (electrophoresis) Treatment of vector with phosphatase Ligation of vector and target sequence with appropriate controls Transformation of competent cells Selection/scoring of recombinants

9 Subcloning and transformation (Selection based on antibiotic resistance) Fig. 25B.4 Fig. 25B.2

10 Sequencing cloned DNA: Sanger method (DNA synthesis + termination chemistry) Generate fragments with a common 5 origin and basespecific 3 termini, each 2,3 ddntp used at 1/10 concentration of dntp mix Fig. 25C.1 or ds plasmid DNA template works too Automated Sanger method involves use of sequencing primer derivatized on the 5 end with either red, blue, green, or yellow fluorescent dyes, plasmid template, and thermostable DNAP (e.g. Taq).

11 Chemical synthesis of a oligonucleotide (non-aqueous solvents, highly reactive phosphoramidites) Pentavalent phosphotriester Dimethoxytrityl, amine blocking groups include benzoyl and isobutyrl Fig. 4B.1

12 Constructing DNA libraries for gene cloning purposes (just buy them!) cdna library (protein-coding region) Purify RNA from tissue of origin: poly(a) RNA Anneal synthetic primer and extend with RT Digest RNA and extend second strand Cleave with restriction enzyme and subclone into λ phage Genomic library (introns, exons, regulatory) Purify DNA and partially digest with 4-base RE Size fractionate DNA to select for long fragments Prepare vector phage and ligate inserts Packaging reactions and transformation Alternative vector: cosmids (modified plasmids carrying cos sequences required for packaging DNA in λ phage particles) Alternative method:pcr-mediated cloning

13 Making cdna:reverse Transcriptases RTs from retroviruses Two enzymatic activities RNA-dependent DNAP RNase H (exo/endo cleavage of RNA component of RNA-DNA hybrids) Commercial enzymes Moloney murine leukemia virus (M-MuLV-RT: weak RNase H activity, preferred for long mrnas, single polypeptide) Avian myeloblastosis virus (AMV-RT, two peptide chains) M-MuLV-RT and AMV-RT differ in ph and temp optima

14 Constructing a cdna library

15 Screening libraries, cells, tissues for genes of interest Probes: cloned or synthetic DNA used to: Evaluate copy number or gene organization Isolate/clone a gene from a library Examine expression of a gene in cells/tissues Sources of probes Synthetic DNA oligonucleotide Same gene but from different species (ortholog) Other related gene (paralog) Methods Hybridization techniques (DNA-DNA, DNA-RNA) Functional screening of expression libraries

16 Hybridization vs functional screening Hybridization Functional induce synthesis of gene products for functional screen (e.g. IPTG) bacterial colonies each with different expressed protein product probe with protein binding partner (e.g. Ab or ligand) Fig. 25B.3

17 Assessing the presence of a gene or its mrna product in a cell or tissue Electrophoresis of restriction fragments of genomic DNA or enriched polya mrna Fig. 25D.1

18 Polymerase Chain Reaction (amplification of a DNA sequence by repetitive cycles of heating, annealing, and extension using ss primers) Basic procedure Heat DNA to separate strands Anneal synthetic oligo primers Extend with Taq polymerase Repeat for cycles Uses Detecting and amplifying a particular target sequence in a large genome Diagnostic tool to detect mutations in certain genes Quantify specific mrna expression (RT-PCR) Site-directed mutagenesis and gene cloning Fig. 24A.1

19 Modification of genes by mutagenesis Primer-assisted in M13 vector (old method) Clone gene into vector single stranded M13 template Add complementary oligonucleotide and extend Transform appropriate strain Select mutants Other site-directed mutagenesis methods Modification by endo or exonuclease digestion (insertion or deletion) Primer or PCR-mediated mutagenesis in dsdna templates Fig. 25E.1

20 but nowadays almost everybody does it this way PfuTurbo DNAP for blunt end polishing Dpn I Figures from Stratagene catalog