DNA Technology and Genetic Engineering

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Dec 12, 2012 (4 years and 8 months ago)

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© 2012 Pearson Education, Inc.

Concepts and Current Issues

Human
Biology

Sixth Edition

Michael D. Johnson

Lecture Presentation

Suzanne Long

Monroe Community College

Chapter 20

DNA Technology and
Genetic Engineering

© 2012 Pearson Education, Inc.

Some Terminology to Get Started


Biotechnology


The technical application of biological
knowledge for human purposes


Recombinant DNA technology


Applied science that explores applications of
cutting, splicing, and creating DNA


Genetic engineering


Manipulation of the genetic makeup of cells or
whole organisms

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DNA Sequencing Reveals DNA Structure


Prior to sequencing, DNA must be
amplified


Primers: enable initiation of DNA
synthesis


A,G,C,T nucleotides


A*, G*, C*, T* fluorescent
-
labeled
dideoxy nucleotides


DNA polymerase: replicates the DNA


Gel electrophoresis: separates DNA
strands by size

© 2012 Pearson Education, Inc.

Figure 20.1

Primer

Sequence to

be analyzed

DNA polymerase

In the first step, short DNA sequences complementary to the sequence

to be analyzed are produced, each ending in a labeled nucleotide.

In the second step, the short DNA sequences produced by the first

step are separated by gel electrophoresis and then analyzed by

a laser.

Complementary

base sequences

A photo of a DNA sequencing gel

The result is a printout of the sequence

of the complementary strand of DNA.

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1

2

3

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DNA Can Be Cloned in the Laboratory


Recombinant DNA


Cutting, splicing, and copying DNA


Tools for manipulating DNA


Restriction enzymes
: cut DNA at specific
sites


DNA ligases
: join fragments of DNA



Plasmids
: small circular pieces of DNA to
which desired genes can be added and
inserted into bacteria for amplification

© 2012 Pearson Education, Inc.

Figure 20.2

Restriction

enzyme cut

Palindromic

sequence

Restriction

enzyme cut

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Figure 20.3

Human cell

E. coli

Plasmid

DNA

Isolate DNA from

bacterial and human cells.

Cut both DNAs with

same restriction enzyme.

Human DNA

containing gene

of interest

Palindromic

sequence

DNAs are mixed. Human

fragments line up with

plasmid by base pairing

of exposed single
-
strand

regions.

DNA fragments

Recombinant

DNA

DNA ligase is added, to

connect human and

plasmid DNA together.

Plasmids are absorbed

by bacteria.

Bacteria containing the

recombinant plasmids of

interest are selected and

cloned.

Bacterial clones carrying copies of the human genes

Bacterial

DNA

1

2

3

4

5

6

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Recombinant DNA

© 2012 Pearson Education, Inc.

The Polymerase Chain Reaction


Can be used to rapidly amplify DNA
sequences to obtain million of copies


DNA to be amplified, primers, heat
-
stable
DNA polymerase are combined


Repeated heating and cooling cycles allow
for rapid amplification of a sequence of DNA
defined by the primers

© 2012 Pearson Education, Inc.

Figure 20.4

Sample of double
-
stranded DNA

DNA is unwound by gentle

heating; the single strands will

serve as templates for new

strands.

The single strands of DNA are

mixed with primers able to bind

to one end. Nucleotides and

DNA polymerase are added.

As the mixture cools, the primers

pair with the ends of the template

strands.

Nucleotides are attached in

sequence, and the DNA strands

replicate. The number of double
-

stranded DNA molecules doubles.

The mixture is reheated,

unwinding the double
-
stranded

DNA molecules again.

The mixture is cooled again.

Primers again pair with the ends

of each strand, and nucleotides

again are attached in sequence.

Replication again doubles the

number of double
-
stranded

DNA molecules.

+

© 2012 Pearson Education, Inc.

The Polymerase Chain
Reaction (PCR)

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Identifying the Source of DNA:

DNA Fingerprinting


Often DNA sample must be amplified by
PCR first


DNA is cut with restriction enzymes


Different individuals will have different
lengths of DNA between the restriction
sites


DNA is separated by gel electrophoresis
and fragment pattern is compared


© 2012 Pearson Education, Inc.

Figure 20.5

Blood Stain

Suspect 1

Suspect 2

Suspect 3

Suspect 4

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Genetic Engineering: Transgenic
Organisms


Transgenic bacteria have been developed
to produce the following


Insulin


Human growth hormone


Erythropoietin


Tissue plasminogen activator (tPA)


Vaccines

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Genetic Engineering: Transgenic
Organisms


Transgenic plants have been modified to
express the following:


Increased resistance to freezing


Longer shelf life


Increased vitamin A


Edible vaccines


Human proteins (i.e., albumin)


Increased resistance to pests

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Transgenic Plants


Concerns


“Frankenfoods”


Plant disease susceptibility could lead to crop
failure

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Transgenic Animals


Bigger challenges


More difficult to introduce foreign DNA to
animal cells


Cloning more difficult


Successes


Bovine growth hormone (bGH): used to
promote faster animal growth


“Gene pharming”: introducing human genes
into dairy animals in such a way that the
human protein is produced in the animal’s
milk

© 2012 Pearson Education, Inc.

Figure 20.10

DNA of

interest

Plasmid

Microinject DNA

into nucleus

Implant into foster

mother

Goat

ovum

Identify transgenic

progeny by PCR

The DNA of interest

is expressed and the

protein is secreted

into the milk

Collect milk

Milk containing

the protein

Separate milk proteins

Isolate the useful protein

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Gene Therapy: Hope of the Future?


Gene therapy


Introduction of human genes into human
cells to treat or correct a disease


Obstacles


Difficult to introduce genes into the “right”
cells where the genes would normally be
expressed


Need effective means of delivering genes


Can corrective genes be introduced into
reproductive cells to stop the passing of
defective genes to offspring?

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Vectors Transfer Genes to Human Cells


Retroviruses


Human genes can be packaged in
retroviruses, which can introduce genes to
human cells


Problems with retroviral vectors


Will only insert genes into dividing cells


Insertion sites are random


Liposomes


Injecting naked DNA


All of these methods are experimental

© 2012 Pearson Education, Inc.

Figure 20.12

Normal gene

Retrovirus

Incorporate normal gene

into retroviral vector

Cell taken

from patient

Infect patient’s cells

with retrovirus vector

Inject retrovirus
-
infected

cells back into patient

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Gene Therapy Successes


Severe combined immunodeficiency
(SCID): some success with gene therapy


Cystic fibrosis: minor improvement


Cancer research

© 2012 Pearson Education, Inc.

GMO