chapter 20 lecture concepts of genetics tenth edition recombinant dna technology
TRANSCRIPT
Chapter 20 Lecture
Concepts of GeneticsTenth Edition
Recombinant DNA Technology
© 2012 Pearson Education, Inc.
20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction
Enzymes and DNA Cloning Vectors
© 2012 Pearson Education, Inc.
Section 20.1
• Recombinant DNA refers to the joining of DNA molecules, usually from different biological sources, that are not found together in nature
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
• Vectors Carry DNA Molecules to Be Cloned
• Lambda () Phage Vectors
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
• Vectors Carry DNA Molecules to Be Cloned
• Cosmid Vectors
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
• Vectors Carry DNA Molecules to Be Cloned
• Bacterial Artificial Chromosomes
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
• Vectors Carry DNA Molecules to Be Cloned
• Expression Vectors
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
• DNA Was First Cloned in Prokaryotic Host Cells
© 2012 Pearson Education, Inc.
Yeast Cells Are Used as Eukaryotic Hosts for Cloning
© 2012 Pearson Education, Inc. Table 13.1
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
• Plant and Animal Cells Can Be Used As Host Cells For Cloning
• Plant Cell Hosts
© 2012 Pearson Education, Inc.
FromAgrobacteriumtumifaciens
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc.
20.2 DNA Libraries Are Collections of Cloned Sequences
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA
© 2012 Pearson Education, Inc. Figure 20.8
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
20.4 Molecular Techniques forAnalyzing DNA
© 2012 Pearson Education, Inc.
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
© 2012 Pearson Education, Inc.
© 2012 Pearson Education, Inc. Figure 20.11
© 2012 Pearson Education, Inc.
© 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
© 2012 Pearson Education, Inc.
20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the
Molecular Level
© 2012 Pearson Education, Inc. Figure 20.15
© 2012 Pearson Education, Inc.
© 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