Chapter 20 Lecture

Concepts of GeneticsTenth Edition

Recombinant DNA Technology

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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

© 2012 Pearson Education, Inc. Figure 20.1

© 2012 Pearson Education, Inc. Figure 20.2

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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)

© 2012 Pearson Education, Inc. Figure 20.3

© 2012 Pearson Education, Inc. Figure 20.4

© 2012 Pearson Education, Inc. Figure 20.5

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• Vectors Carry DNA Molecules to Be Cloned

• Lambda () Phage Vectors

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• Vectors Carry DNA Molecules to Be Cloned

• Cosmid Vectors

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• Vectors Carry DNA Molecules to Be Cloned

• Bacterial Artificial Chromosomes

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• Vectors Carry DNA Molecules to Be Cloned

• Expression Vectors

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• DNA Was First Cloned in Prokaryotic Host Cells

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Yeast Cells Are Used as Eukaryotic Hosts for Cloning

© 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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FromAgrobacteriumtumifaciens

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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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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

© 2012 Pearson Education, Inc. Figure 20.6

© 2012 Pearson Education, Inc. Figure 20.7

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20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA

© 2012 Pearson Education, Inc. Figure 20.8

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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

© 2012 Pearson Education, Inc. Figure 20.9

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20.4 Molecular Techniques forAnalyzing 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

© 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.

© 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

© 2012 Pearson Education, Inc. Figure 20.14

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20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the

Molecular Level

© 2012 Pearson Education, Inc. Figure 20.15

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© 2012 Pearson Education, Inc.Figure 19-27 Copyright © 2006 Pearson Prentice Hall, Inc.

TTCGTG

AA 5’-TTCGTGAA…etc

© 2012 Pearson Education, Inc. Figure 20.16

© 2012 Pearson Education, Inc. Figure 20.17

© 2012 Pearson Education, Inc. Figure 20.18

© 2012 Pearson Education, Inc. Figure 20.19