powerpoint lecture presentations prepared by bradley...

PowerPoint® Lecture

Presentations prepared by

Bradley W. Christian,

McLennan Community

College

C H A P T E R

© 2016 Pearson Education, Ltd.

Biotechnology and DNA Technology

9


Introduction to Biotechnology

• Biotechnology: the use of microorganisms, cells, or cell components to make a product

• Foods, antibiotics, vitamins, enzymes

• Recombinant DNA (rDNA) technology: theinsertion or modification of genes to produce desired proteins


An Overview of Recombinant DNA Procedures

• Vector: self-replicating DNA molecule used to transport foreign DNA into a cell

• Clone: population of genetically identical cells arising from one cell; each carries the vector


Figure 9.1 A Typical Genetic Modification Procedure.


Tools of Biotechnology

• Selection: selecting for a naturally occurring microbe that produces a desired product

• Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait

• Site-directed mutagenesis: a targeted and specific change in a gene


Restriction Enzymes

• Cut specific sequences of DNA

• Destroy bacteriophage DNA in bacterial cells

• Methylated cytosines in bacteria protect their own DNA from digestion

• Create blunt ends or staggered cuts known as sticky ends


Recombinant DNA Technology

PLAY Animation: Recombinant DNA Technology

recombinant_dna.html


Table 9.1 Selected Restriction Enzymes Used in rDNA Technology


Figure 9.2 A restriction enzyme's role in making rDNA.

A restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites, shown in blue.

These cuts produce a DNA fragment with

two sticky ends.

DNA from another source,

perhaps a plasmid, cut with

the same restriction enzyme

When two such fragments of DNA cut

by the same restriction enzyme come

together, they can join by base pairing.

The joined fragments will usually form either a linear

molecule or a circular one, as shown here for a plasmid.

Other combinations of fragments can also occur.

The enzyme DNA ligase is used to unite the

backbones of the two DNA fragments,

producing a molecule of rDNA.

Recognition sites

DNA Cut Cut

Cut Cut

Sticky end

rDNA


Vectors

• Carry new DNA to desired cells

• Must be able to self-replicate

• Plasmids and viruses can be used as vectors

• Shuttle vectors exist in several different species and can move cloned sequences among various organisms


Figure 9.3 A plasmid used for cloning.

pUC19ampR

lacZ

HindIIIBamHIEcoRI

ori


Polymerase Chain Reaction

• Process of increasing small quantities (amplifying) of DNA for analysis

• Used for diagnostic tests for genetic diseases and detecting pathogens

• Reverse-transcription PCR uses mRNA as template


PCR: Overview

PLAY Animation: PCR: Overview

pcr_polymerase.html


PCR: Components

PLAY Animation: PCR: Components

pcr_components.html


Figure 9.4 The polymerase chain reaction.


Inserting Foreign DNA into Cells

• DNA can be inserted into a cell by:

• Transformation: Cells take up DNA from the surrounding environment

• Electroporation: Electrical current forms pores in cell membranes

• Protoplast fusion: Removing cell walls from two bacteria allows them to fuse


Chromosome

Plasma membrane

Cell wall

Bacterial cell walls

are enzymatically

digested, producing

protoplasts.

Bacterial cells

Protoplasts

In solution, protoplasts are

treated with polyethylene glycol.

Protoplasts fuse.

Segments of the two

chromosomes recombine.

Recombinant cell

grows new cell wall.

Recombinant

cell

Figure 9.5 Protoplast fusion.


Inserting Foreign DNA into Cells

• DNA can be inserted into a cell by:

• Gene gun

• Microinjection


Figure 9.7 The microinjection of foreign DNA into an egg.


Genomic Libraries

• Collections of clones containing different DNA fragments

• An organism's DNA is digested and spliced into plasmid or phage vectors and introduced into bacteria

• At least one clone exists for every gene in the organism


Figure 9.8 Genomic libraries.

Genome to be stored

in library is cut up with

restriction enzyme

Host

cell

Recombinant

plasmidOR

Recombinant

phage DNA

Phage cloning

vector

Plasmid Library Phage Library


Genomic Libraries

• Complementary DNA (cDNA) is made from mRNA by reverse transcriptase

• Used for obtaining eukaryotic genes because eukaryotic DNA has introns that do not code for protein

• mRNA has the introns removed, coding only for the protein product


Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene.

Exon Intron IntronExon Exon

DNA

Nucleus

A gene composed of exons and

introns is transcribed to RNA by

RNA polymerase.

RNA

transcript

Processing enzymes in the nucleus

remove the intron-derived RNA

and splice together the

exon-derived RNA into mRNA.

mRNA

Cytoplasm

mRNA is isolated from the

cell, and reverse

transcriptase is added.

First strand

of DNA is

synthesized.

The mRNA is digested by

reverse transcriptase.

DNA strand

being synthesized

DNA polymerase is added to

synthesize second strand

of DNA.

cDNA of

gene without

introns Test tube


Synthetic DNA

• Builds genes using a DNA synthesis machine


Selecting a Clone

• Blue-white screening

• Uses plasmid vector containing ampicillin resistance gene (ampR) and β-galactosidase gene (lacZ)

• Bacteria is grown in media containing ampicillin and X-gal, a substrate for β-galactosidase


Figure 9.11 Blue-white screening, one method of selecting recombinant bacteria.

Ampicillin-resistance

gene (ampR)

Plasmid DNA and foreign DNAare both cut with the same

restriction enzyme. The plasmidhas the genes for lactose hydrolysis(the lacZ gene encodes the enzyme

β-galactosidase) and ampicillinresistance.

Plasmid

Foreign DNA

Recombinant

plasmidForeign DNA will insert intothe lacZ gene. The bacterium

receiving the plasmid vectorwill not produce the enzymeβ-galactosidase if foreign

DNA has been inserted intothe plasmid.

The recombinant plasmidis introduced into a

bacterium, which becomesampicillin resistant.

All treated bacteria are spreadon a nutrient agar plate

containing ampicillin and aβ-galactosidase substrate andincubated. The β-galactosidase

substrate is called X-gal.

Only bacteria that picked upthe plasmid will grow in the

presence of ampicillin.Bacteria that hydrolyze X-gal produce galactose and

an indigo compound. The indigo turns the colonies blue. Bacteria that cannot hydrolyze

X-gal produce white colonies.

Bacterium

Restriction

site

β-galactosidase gene (lacZ)

Restriction

sites

Colonieswith foreign

DNA


Selecting a Clone

• Colony hybridization

• Use DNA probes: short segments of single-stranded DNA complementary to the desired gene


Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest.

Make replica of master

plate on nitrocellulose

filter.

Treat filter with

detergent (SDS)

to lyse bacteria.

Treat filter with

sodium hydroxide

(NaOH) to separate

DNA into single

strands.

Bound

DNA

probe

Replica plate

Compare filter with

replica of master plate

to identify colonies

containing gene of

interest.

Wash filter to

remove unbound

probe.

Probe will hybridize

with desired gene

from bacterial cells.

Fluorescence

labeled probes

Add labeled probes.

Nitrocellulose

filter

Strands of

bacterial DNA

Master plate with

colonies of bacteria

containing cloned

segments of foreign

genes

Gene of

interest

Single-

stranded

DNA

Colonies containing

genes of interest


Making a Gene Product

• E. coli• Advantages: easily grown and its genomics are known

• Disadvantages: produces endotoxins and does not secrete its protein products


Making a Gene Product

• Saccharomyces cerevisiae• Easily grown and has a larger genome than bacteria

• Expresses eukaryotic genes easily

• Plant cells and whole plants

• Express eukaryotic genes easily

• Plants are easily grown, large-scale, and low-cost

• Mammalian cells

• Express eukaryotic genes easily

• Can make products for medical use

• Harder to grow


Therapeutic Applications

• Human enzymes and other proteins such as insulin

• Subunit vaccines: made from pathogen proteins in genetically modified yeasts

• Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines

• Gene therapy to replace defective or missing genes


Table 9.2 Some Pharmaceutical Products of rDNA (1 of 2)


Table 9.2 Some Pharmaceutical Products of rDNA (2 of 2)


Therapeutic Applications

• Gene silencing

• Small interfering RNAs (siRNAs) bind to mRNA, which is then destroyed by RNA-induced silencing complex (RISC)

• RNA interference (RNAi) inserts DNA encoding siRNA into a plasmid and transferred into a cell


Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.


Genome Projects

• Shotgun sequencing sequences small pieces of genomes which are assembled by a computer

• Metagenomics is the study of genetic material directly from environmental samples

• The Human Genome Project sequenced the entire human genome

• The Human Proteome Project will map proteins expressed in human cells


Figure 9.15 Shotgun sequencing.

Isolate DNA.

Fragment DNA

with restriction

enzymes.

Clone DNA

in a bacterial

artificial

chromosome

(BAC).

Construct a gene library Random sequencing Closure phase

Sequence DNA fragments.

Edit

sequences;

fill in gaps.

Assemble sequences.


Scientific Applications

• Bioinformatics: understanding gene function via computer-assisted analysis

• Proteomics: determining proteins expressed in a cell

• Reverse genetics: discovering gene function from a genetic sequence


Scientific Applications

• Southern blotting: DNA probes detect specific DNA in fragments (RFLPs) separated by gelelectrophoresis


Figure 9.16 Southern blotting.

Restriction enzyme

DNA containing the gene of interest is extracted from

human cells and cut into fragments by restriction

enzymes. Fragments are called restriction fragment

length polymorphisms, or RFLPs (pronounced "rif-lips").

The DNA bands are transferred to a nitrocellulose filter by

blotting. The solution passes through the gel and filter to

the paper towels by capillary action.

Labeled probes

This produces a nitrocellulose filter with DNA fragments

positioned exactly as on the gel.

The fragments are separated according to size by gel

electrophoresis. Each band contains many copies of a

particular DNA fragment. The bands are invisible but can

be made visible by staining.

Smaller

Larger

Gene of interest

The filter is exposed to a labeled probe for a specific gene.

The probe will base-pair (hybridize) with a short sequence

present on the gene.

The fragment containing the gene of interest is identified by a

band on the filter.

Gel

Human

DNA

fragments

Nitrocellulose

filter

DNA

transferred

to filter

Nitrocellulose

filter

Gel

Sponge

Paper towels

Salt solution

Gel

Sealable

plastic bag


Forensic Microbiology

• DNA fingerprinting is used to identify pathogens

• PCR microarrays and DNA chips can screen samples for multiple pathogens

• Differs from medicine because it requires:

• Proper collection of evidence

• Establishing a chain of custody


Figure 9.17 DNA fingerprints used to track an infectious disease.

E. coli isolates from

patients whose

infections were

not juice related

Apple juice

isolates

E. coli isolates from

patients who drank

contaminated juice


Nanotechnology

• Bacteria can make molecule-sized particles

• Nanospheres used in drug targeting and delivery


Agricultural Applications

• Ti plasmid: occurs in Agrobacterium tumefaciens• Integrates into the plant genome and causes a

tumorlike growth

• Can be used to introduce rDNA into a plant


Figure 9.19 Crown gall disease on a rose plant.

Crown gall


Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants.

Agrobacterium tumefaciens

bacterium

Ti

plasmid

The plasmid is removed

from the bacterium, and

the T-DNA is cut by a

restriction enzyme.

The plasmid

is reinserted

into a bacterium.

Recombinant

Ti plasmid

The foreign DNA is

inserted into the T-DNA

of the plasmid.

Foreign DNA is cut

by the same enzyme.

A plant is generated from a cell

clone. All of its cells carry the

foreign gene and may express

it as a new trait.

The plant cells

are grown in

culture.

The bacterium is

used to insert the

T-DNA carrying the

foreign gene into the

chromosome of a

plant cell.

Inserted T-DNA

carrying foreign

gene

Restriction

cleavage

site

T-DNA


Agricultural Applications

• Bt toxin

• Herbicide resistance

• Suppression of genes

• Antisense DNA

• Nutrition

• Human proteins


Table 9.3 Some Agriculturally Important Products of rDNA Technology


Safety Issues and Ethics of Using DNA Technology• Need to avoid accidental release into the

environment

• Genetically modified crops must be safe for consumption and for the environment

• Who will have access to an individual's genetic information?

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