DNA TechnologyCh. 20

Figure 20.1 An overview of how bacterial plasmids are used to clone genes

DNA Technology

• Recombinant DNA technology– Set of techniques for recombining

genes from different sources in vitro and transferring the recombinant DNA to a cell where it is expressed

– Typically uses a plasmid as its vector

– Same restriction enzymes used to make “sticky end” cuts

Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

Action of Restriction Enzymes

Restriction Enzymes

• Cut DNA after specific base sequences = restriction site

• Protect bacteria– cut up foreign DNA

• Sticky ends– jagged cut so other DNA can join

• DNA ligase– makes addition of DNA permanent

DNA Technology

• Biotechnology– Refers to the use of living organisms

or components to do practical tasks– Wine industry– Cheese industry– Selective breeding of livestock and

crops– Production of antibiotics

Cloning a Eukaryotic Gene

• Isolation of vector & gene-source DNA– Cloning vector is the original plasmid

• Insertion of DNA into vector– Use of restriction enzymes– May need to make cell competent (E. coli)

• Introduction of cloning vector into cells– Naked DNA added to culture– Bacteria take in plasmid by

transformation

Cloning a Eukaryotic Gene

• Cells reproduce in a culture– Transformed cells are producing new

cells with the cloned gene

• Identification of cell clones– Typically use the R plasmid for ampicillin

resistance– Only cells that have transformed can

grow on the ampicillin agar– Nucleic acid hybridization

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 1)

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 2)

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

Figure 20.4 Using a nucleic acid probe to identify a cloned gene

Chapter 13

Polymerase Chain Reaction

Section 1 DNA Technology

Polymerase Chain Reaction• Use when source of DNA is impure or

scarce• Clones DNA entirely in vitro• Making many copies of a specific segment

of DNA (billions of copies in a few hours)• Used for DNA analysis

– Ancient DNA fragments– DNA from tiny samples– DNA from single embryonic cells– DNA of viral genes

Polymerase Chain Reaction

• Devised in 1985

• Starting materials:– DNA polymerase, primers, nucleotides

Polymerase Chain Reaction

• Heat to separate DNA strands– use DNA polymerase from a bacteria

that lives in hot springs

• Cool to allow primers to bind

• DNA polymerase extends the 3’ end of each primer

• Multiplies exponentially

Southern Blots• Hybridization technique that enables

researchers to determine the presence of certain nucleotide sequences in a sample of DNA

• RFLP’s– differences in DNA sequence on homologous

chromosomes that result in different patterns of restriction fragment lengths for every species

– useful as genetic markers– Inherited following Mendel’s patterns

Southern Blots

• Combination of 5 techniques– Restriction fragment preparation

– Electrophoresis

– Blotting (DNA bands transferred to nitrocellulose paper)

– Hybridization with radioactive probe (attach to gene of interest)

– Autoradiography

Chapter 13

Gel Electrophoresis

Section 1 DNA Technology

Chapter 13

DNA Fingerprint

Section 1 DNA Technology

Figure 20.8 Gel electrophoresis of macromolecules

Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

Figure 20.10 Restriction fragment analysis by Southern blotting

Practical Applications• Diagnosis

– early detection of disease before symptoms show or even birth

– use probes with cloned genes

• Human gene therapy– traceable genetic disorders– may eventually be correctable– replace defective genes with functional genes– only effective if cells receiving normal allele

rapidly reproduce

Figure 20.16 One type of gene therapy procedure

Practical Applications

• Environmental– microorganisms to get rid of waste

– mining

– recycling of wastes and detoxifying

– sewage treatment plants

Practical Applications• Pharmaceutical products

– insulin, growth hormone

• Forensics– blood and tissue type

– RFLP and Southern Blots

• Agricultural– animal husbandry

– cellulase

Figure 20.17 DNA fingerprints from a murder case

Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

Figure 20.x1a Laboratory worker reviewing DNA band pattern

Figure 20.x1b DNA study in CDC laboratory