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Chapter 20 Lecture
Concepts of Genetics
Tenth Edition
Recombinant DNA
Technology
20.1 Recombinant DNA Technology
Began with Two Key Tools: Restriction
Enzymes and DNA Cloning Vectors
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Section 20.1
• Recombinant DNA refers to the joining of
DNA molecules, usually from different
biological sources, that are not found
together in nature
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Section 20.1
• The basic procedure for producing
recombinant DNA involves
– generating specific DNA fragments using
restriction enzymes
– joining these fragments with a vector
– transferring the recombinant DNA molecule to
a host cell to produce many copies that can
be recovered from the host cell
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Section 20.1
• The recovered copies of a recombinant
DNA molecule are referred to as clones
and can be used to study the structure and
orientation of the DNA
• Recombinant DNA technology is used to
isolate, replicate, and analyze genes
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Figure 20.1
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Figure 20.2
Section 20.1
• Vectors are carrier DNA molecules that
can replicate cloned DNA fragments in a
host cell
• Vectors must be able to replicate
independently and should have several
restriction enzyme sites to allow insertion
of a DNA fragment
• Vectors should carry a selectable gene
marker to distinguish host cells that have
taken them up from those that have not
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Section 20.1
• A plasmid is an extrachromosomal
double-stranded DNA molecule that
replicates independently from the
chromosomes within bacterial cells
(Figure 20.3a)
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© 2012 Pearson Education, Inc.
Figure 20.3
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Figure 20.4
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Figure 20.5
•
Vectors Carry DNA Molecules to Be
Cloned
•
Lambda () Phage Vectors
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© 2012 Pearson Education, Inc.
•
Vectors Carry DNA Molecules to Be
Cloned
•
Cosmid Vectors
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© 2012 Pearson Education, Inc.
•
•
Vectors Carry DNA Molecules to Be
Cloned
Bacterial Artificial Chromosomes
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© 2012 Pearson Education, Inc.
•
Vectors Carry DNA Molecules to Be
Cloned
•
Expression Vectors
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© 2012 Pearson Education, Inc.
•
DNA Was First Cloned in Prokaryotic
Host Cells
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Yeast Cells Are Used as Eukaryotic
Hosts for Cloning
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© 2012 Pearson Education, Inc.
Table 13.1
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•
Plant and Animal Cells Can Be Used
As Host Cells For Cloning
•
Plant Cell Hosts
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From
Agrobacterium
tumifaciens
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Section 20.1
• A variety of different human cell types can
be grown in culture and used to express
genes and proteins
• These lines can be subjected to various
approaches for gene or protein functional
analysis, including drug testing for
effectiveness at blocking or influencing a
particular recombinant protein being
expressed, especially if the cell lines are of
a human disease condition such as cancer
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© 2012 Pearson Education, Inc.
20.2 DNA Libraries Are Collections of
Cloned Sequences
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Section 20.2
• DNA libraries represent a collection of cloned
DNA samples derived from a single source that
could be a particular tissue type, cell type, or
single individual
• A genomic library contains at least one copy of
all the sequences in the genome of interest
• Genomic libraries are constructed by cutting
genomic DNA with a restriction enzyme and
ligating the fragments into vectors, which are
chosen depending on the size of the genome
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Section 20.2
• Complementary DNA (cDNA) libraries
contains complementary DNA copies
made from the mRNAs present in a cell
population and represents the genes that
are transcriptionally active at the time the
cells were collected for mRNA isolation
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© 2012 Pearson Education, Inc.
Figure 20.6
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Figure 20.7
20.3 The Polymerase Chain Reaction Is a
Powerful Technique for Copying DNA
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© 2012 Pearson Education, Inc.
Figure 20.8
Section 20.3
• Reverse transcription PCR (RT-PCR) is
used to study gene expression by studying
mRNA production by cells or tissues
• Quantitative real-time PCR (qPCR) or
real-time PCR allows researchers to
quantify amplification reactions as they
occur in ‘real time’ (Figure 20.9)
– The procedure uses an SYBR green dye and
TaqMan probes, which contain two dyes
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© 2012 Pearson Education, Inc.
Figure 20.9
20.4 Molecular Techniques for
Analyzing DNA
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Section 20.4
• A restriction map establishes the number
and order of restriction sites and the
distance between restriction sites on a
cloned DNA segment
• It provides information about the length of
the cloned insert and the location of
restriction sites within the clone
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© 2012 Pearson Education, Inc.
Figure 20.10
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© 2012 Pearson Education, Inc.
Figure 20.11
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© 2012 Pearson Education, Inc.
Section 20.4
• Northern blot analysis is used to
determine whether a gene is actively being
expressed in a given cell or tissue
– Used to study patterns of gene expression in
embryonic tissues, cancer, and genetic
disorders
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© 2012 Pearson Education, Inc.
Figure 20.14
20.5 DNA Sequencing Is the Ultimate Way
to Characterize DNA Structure at the
Molecular Level
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© 2012 Pearson Education, Inc.
Figure 20.15
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A
A
G
T
CG
TT
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Figure 19-27
5’-TTCGTGAA…etc
Copyright © 2006 Pearson Prentice Hall, Inc.
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Figure 20.16
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Figure 20.17
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Figure 20.18
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Figure 20.19