Chapter 8: Recombinant DNA. Ways this technology touches us. Overview. Genetic Engineering

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1 Chapter 8 Recombinant DNA and Genetic Engineering Genetic manipulation Ways this technology touches us Criminal justice The Justice Project, started by law students to advocate for DNA testing of Death Row Inmates, led the governor of Illinois to stop all executions Immigration claims of family status (children, parents of legal immigrants) for granting visas upheld or rejected by DNA tests Tailored health care- genetic medicine MIAs in armed forces Overview Recombinant DNA technology exploits features of genes, gene expression, and DNA enzymology to create novel DNA molecules for study. Foreign DNA is spliced into a vector for amplification, producing a clone of the inserted DNA. Restriction endonucleases cut DNA at specific target sites. Polymerase chain reaction (PCR) can be used for specific DNA amplification. Labeled single-stranded DNA or RNA can be used as a probe to identify molecules containing its base-pair complement. Virtually any nucleotide sequence, including restriction sites can be mapped. DNA can be sequenced. Transgenes can be constructed and expressed in foreign hosts.

2 Recombinant DNA technology Active growth since the mid-1970s Genetic engineering applies recombinant DNA technology to problems in biology, medicine, and agriculture Genomics studies information contained in the genome at the molecular level Made possible by: ability of single-stranded polynucleotides to base pair with their complement ability of proteins to recognize target DNA sequences ability to join DNA molecules together, creating recombinant DNA ability to amplify any DNA molecule: cloning Generating recombinant DNA (1) DNA sources genomic DNA from chromosomes usually too large to clone directly cdna (complementary DNA) derived by action of reverse transcriptase from (usually) mrna template chemically synthesized oligonucleotides Digestion of DNA by restriction enzyme recognize palindromic double-stranded sequences produce complementary sticky ends 5 -G^AATTC-3 dozens of such enzymes 3 -CTTAA^G-5 sticky ends can be ligated under appropriate conditions

3 Generating recombinant DNA (2) Insertion into vector cloning vectors permit replication of inserted DNA include plasmids, vectors, artificial chromosomes complementary restriction ends joined by DNA ligase multiple fragments can be joined Transformation into expression system bacterial cell, e.g., E. coli eukaryotic cell, e.g., yeast

4 Polymerase chain reaction (PCR) Must know sequences flanking desired region No cloning procedures necessary Principle: DNA made in one amplification cycle is used as template in subsequent cycle heat denaturation to yield single-stranded DNA annealing of primers (oligonucleotides) to singlestranded DNA extension of primers by thermostable DNA polymerase Highly sensitive, requiring as little as one copy of single-stranded DNA as initial template PCR summary Works best for amplification of fragments bp long Highly subject to environmental contamination : good lab practices essential Possible artifacts include jumping, non-specific amplification (pseudogenes, other members of gene family Some sequence information must be known already to synthesize primers New innovations reported using Helicase instead of DNA polymerase (don t need a thermocycler!)

5 Cloning in host cell Requires use of cloning vector or artificial chromosome Recombinant molecule must enter host cell Recombinant molecule must efficiently replicate Replicated recombinant molecule must be recovered from host clone Many commercial products available Cloning vectors Common properties origin of DNA replication unique restriction sites for insertion of DNA multiple cloning sites containing many restriction sites engineered into many plasmid vectors Types of vectors plasmids containing drug resistance gene many commercially available plasmids bacteriophage, e.g., lambda cosmids for larger DNA molecules BAC: bacterial artificial chromosome YAC: yeast artificial chromosome