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Chapter 20 Lecture Concepts of Genetics Tenth Edition Recombinant DNA Technology.

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Presentation on theme: "Chapter 20 Lecture Concepts of Genetics Tenth Edition Recombinant DNA Technology."— Presentation transcript:

1 Chapter 20 Lecture Concepts of Genetics Tenth Edition Recombinant DNA Technology

2 © 2012 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors

3 © 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

4 © 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

5 © 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

6 © 2012 Pearson Education, Inc. Figure 20.1

7 © 2012 Pearson Education, Inc. Figure 20.2

8 © 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

9 © 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)

10 © 2012 Pearson Education, Inc. Figure 20.3

11 © 2012 Pearson Education, Inc. Figure 20.4

12 © 2012 Pearson Education, Inc. Figure 20.5

13 © 2012 Pearson Education, Inc. Vectors Carry DNA Molecules to Be Cloned Lambda ( ) Phage Vectors

14 © 2012 Pearson Education, Inc.

15 Vectors Carry DNA Molecules to Be Cloned Cosmid Vectors

16 © 2012 Pearson Education, Inc.

17 Vectors Carry DNA Molecules to Be Cloned Bacterial Artificial Chromosomes

18 © 2012 Pearson Education, Inc.

19 Vectors Carry DNA Molecules to Be Cloned Expression Vectors

20 © 2012 Pearson Education, Inc.

21 DNA Was First Cloned in Prokaryotic Host Cells

22 © 2012 Pearson Education, Inc. Yeast Cells Are Used as Eukaryotic Hosts for Cloning

23 © 2012 Pearson Education, Inc. Table 13.1

24 © 2012 Pearson Education, Inc.

25 Plant and Animal Cells Can Be Used As Host Cells For Cloning Plant Cell Hosts

26 © 2012 Pearson Education, Inc. From Agrobacterium tumifaciens

27 © 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

28 © 2012 Pearson Education, Inc.

29 20.2 DNA Libraries Are Collections of Cloned Sequences

30 © 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

31 © 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

32 © 2012 Pearson Education, Inc. Figure 20.6

33 © 2012 Pearson Education, Inc. Figure 20.7

34 © 2012 Pearson Education, Inc. 20.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA

35 © 2012 Pearson Education, Inc. Figure 20.8

36 © 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

37 © 2012 Pearson Education, Inc. Figure 20.9

38 © 2012 Pearson Education, Inc. 20.4 Molecular Techniques for Analyzing DNA

39 © 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

40 © 2012 Pearson Education, Inc. Figure 20.10

41 © 2012 Pearson Education, Inc.

42 Figure 20.11

43 © 2012 Pearson Education, Inc.

44

45 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

46 © 2012 Pearson Education, Inc. Figure 20.14

47 © 2012 Pearson Education, Inc. 20.5 DNA Sequencing Is the Ultimate Way to Characterize DNA Structure at the Molecular Level

48 © 2012 Pearson Education, Inc. Figure 20.15

49 © 2012 Pearson Education, Inc.

50 Figure 19-27 Copyright © 2006 Pearson Prentice Hall, Inc. T T C G T G A A 5’-TTCGTGAA…etc

51 © 2012 Pearson Education, Inc. Figure 20.16

52 © 2012 Pearson Education, Inc. Figure 20.17

53 © 2012 Pearson Education, Inc. Figure 20.18

54 © 2012 Pearson Education, Inc. Figure 20.19


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