The Role of Recombinant DNA Technology in Biotechnology Intentional modification of organisms’ genomes for practical purposes Three goals Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need © 2012 Pearson Education Inc. 1

Figure 8.1 Overview of recombinant DNA technology Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid Isolate plasmid. Gene of interest Enzymatically cleave DNA into fragments. Isolate fragment with the gene of interest. Insert gene into plasmid. Insert plasmid and gene into bacterium. Culture bacteria. Harvest copies of gene to insert into plants or animals Harvest proteins coded by gene Eliminate undesirable phenotypic traits Create beneficial combination of traits Produce vaccines, antibiotics, hormones, or enzymes

Plasmids A plasmid is a circular, self-replicating DNA molecule carrying a few, useful but non necessary genes. Occurence prokaryote organisms eukaryotic organisms like Entamoeba histolytica, yeast etc. Their size varies from 1 kbp to over 400 kilobase pairs (kbp). In a single cell there are anywhere from one copy, for large plasmids, to hundreds of copies of the same plasmid.

We speaks of low and high copy number plasmids Plasmids are easy to manipulate and isolate from bacteria (kits). After being modified, they can be integrated into other genomes, plants, protists, mammals, thereby conferring to other organisms whatever genetic functionality they carry. Thus, this gives the ability to introduce genes into a given organism by using bacteria to amplify the hybrid genes that are created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.

Bacterial Conjugation

Plasmids in Applied molecular biology Minimum requirements for plasmids useful for recombination technology: 1. Origin of replication (ORI). ORI enables a plasmid DNA to be duplicated independently from the chromosome 2. Selectable marker: allow to select for cells that have your plasmids. 3. Restriction enzyme sites in non-essential regions of the plasmid. Plasmid replication initiates in a cis-site called ori. It proceeds either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

Plasmids often contain genes or gene-cassettes that confer a selective advantage when they are inside a bacterium: resistance to antibiotics resistance to herbicides insecticide production to the bacterium harboring them, for example, the ability to make the bacterium antibiotic resistant.

The Tools of Recombinant DNA Technology Mutagens Physical and chemical agents that produce mutations Scientists utilize mutagens to Create changes in microbes’ genomes to change phenotypes Select for and culture cells with beneficial characteristics Mutated genes alone can be isolated © 2012 Pearson Education Inc. 8

The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize cDNA Isolated from retroviruses Uses RNA template to transcribe molecule of cDNA Easier to isolate mRNA molecule for desired protein first mRNA of eukaryotes has introns removed Allows cloning in prokaryotic cells © 2012 Pearson Education Inc. 9

The Tools of Recombinant DNA Technology Synthetic Nucleic Acids Molecules of DNA and RNA produced in cell-free solutions Uses of synthetic nucleic acids Elucidating the genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate specific sequences of nucleotides Synthesizing antisense nucleic acid molecules © 2012 Pearson Education Inc. 10

The Tools of Recombinant DNA Technology Restriction Enzymes Bacterial enzymes that cut DNA molecules only at restriction sites Categorized into two groups based on type of cut Cuts with sticky ends Cuts with blunt ends © 2012 Pearson Education Inc. 11

Figure 8.2 Actions of restriction enzymes-overview

The Tools of Recombinant DNA Technology Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids © 2012 Pearson Education Inc. 13

Figure 8.3 Producing a recombinant vector mRNA for human growth hormone (HGH) Antibiotic resistance gene Restriction site Reverse transcription cDNA for HGH Plasmid (vector) Restriction enzyme Restriction enzyme Sticky ends Gene for human growth hormone Ligase Recombinant plasmid Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.

The Tools of Recombinant DNA Technology Gene Libraries A collection of bacterial or phage clones Each clone in library often contains one gene of an organism’s genome Library may contain all genes of a single chromosome Library may contain set of cDNA complementary to mRNA © 2012 Pearson Education Inc. 15

Figure 8.4 Production of a gene library-overview Genome Isolate genome or organism. Generate fragments using restriction enzymes. Insert each fragment into a vector. Introduce vectors into cells. Culture recombinant cells; descendants are clones.

Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Large number of identical molecules of DNA produced in vitro Critical to amplify DNA in variety of situations Epidemiologists use to amplify genome of unknown pathogen Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores © 2012 Pearson Education Inc. 17

Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Repetitive process consisting of three steps Denaturation Priming Extension Can be automated using a thermocycler © 2012 Pearson Education Inc. 18

Figure 8.5a The use of PCR to replicate DNA, steps 1-3 Original DNA molecule 3´ 3´ 5´ 5´ Heat to 94°C Denaturation DNA primer Deoxyribonucleotide triphosphates Priming DNA polymerase Cool to 65°C DNA polymerase 3´ 5´ 5´ Extension DNA primer 5´ 5´ 3´ 72°C

Figure 8.5b The use of PCR to replicate DNA, step 4 First cycle Second cycle Third cycle Fourth cycle 2 DNA molecules 4 DNA molecules 8 DNA molecules Repeat 16 DNA molecules