1. 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

2. 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

3. 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.

4. 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.

5. Bacterial Conjugation

6. 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.

7. 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.

8. 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

9. 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

10. 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

11. 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

12. Figure 8.2 Actions of restriction enzymes-overview

13. 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

14. 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.

15. 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

16. 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.

17. 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

18. 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

19. 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

20. 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