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Page 1: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

CHAPTER 20DNA TECHNOLOGY AND GENOMICS

Page 2: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same molecule.

Introduction

Page 3: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

DNA technology makes it possible to clone genes for basic research and commercial applications: an overview

Page 4: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Restriction enzymes- cut DNA molecules at specific locations.

• In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria.

• Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences.– Bacteria protect their own DNA by methylation.

Restriction enzymes are used to make recombinant DNA

Page 5: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Each restriction enzyme cleaves a specific sequence of bases or restriction site.– These are often a symmetrical series of four to

eight bases on both strands running in opposite directions.• If the restriction site on one strand is 3’-CTTAGG-5’, the

complementary strand is 5’-GAATTC-3’.

• Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.– Copies of a DNA molecule will always yield the

same set of restriction fragments when exposed to a specific enzyme.

Page 6: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Restriction enzymes cut in a staggered way creating single-stranded ends, sticky ends.– These extensions will form hydrogen-bonded base

pairs with complementary single-stranded stretches on other DNA molecules cut with the same restriction enzyme.

• These DNA fusions can be made permanent by DNA ligase which seals the strand by catalyzing the formation of phosphodiester bonds.

Page 7: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Restriction enzymes and DNA ligase can be used to make recombinant DNA, DNA that has been spliced together from two different sources.

Page 8: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

Genes can be cloned in DNA vectors: a closer look

Page 9: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Yeast artificial chromosomes (YACs) - an origin site for replication, a centromere, and two telomeres, can carry foreign DNA up to a million bp long.

• Bacterial artificial chromosome (BAC)- can carry inserts of 100,000 to 500,000 base pairs.

Page 10: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• This technique can quickly amplify any piece of DNA without using cells.

• In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules.

• The key to easy PCR automation was the discovery of an unusual DNA polymerase, isolated from bacteria living in hot springs, which can withstand the heat needed to separate the DNA strands at the start of each cycle.

The polymerase chain reaction (PCR) clones DNA entirely in vitro

Page 11: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Taq polymerase- isolated from Thermus aquaticus

• Lacks a 3’ to 5’ exonuclease proofreading ability. Error rate of 1 in 9,000 bp

• Kary Mullis, Cetus Corporation, noble prize 1993

• Hoffman-La Rohce bought Taq patents for $330M, probably generated $2B in revenue

Page 12: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same
Page 13: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• PCR has amplified DNA from a variety of sources:– fragments of ancient DNA from a 40,000-year-old

frozen wooly mammoth,– DNA from tiny amount of blood or semen found at

the scenes of violent crimes,– DNA from single embryonic cells for rapid prenatal

diagnosis of genetic disorders,– DNA of viral genes from cells infected with difficult-

to-detect viruses such as HIV.

Page 14: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

DNA Analysis and Genomics• Gel electrophoresis separates macromolecules -

nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field.

• For linear DNA molecules, separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel.

Page 15: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same
Page 16: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Southern Blotting

Page 17: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Restriction fragment length polymorphisms (RFLPs)

• DNA is cut with restriction enzymes. Because DNA differs from person to person, where the restriction enzyme cuts will differ. Different length fragments will be generated depending on the DNA sequence.

• Single nucleotide polymorphisms (SNPs)– Most of our diversity is in the form of single base-

pair variations.• In humans, SNPs occur about once in 1,000 bases, meaning

that any two humans are 99.9% identical.

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• Human Genome Project, begun in 1990.– This is an effort to map the entire human genome,

ultimately by determining the complete nucleotide sequence of each human chromosome.

– An international, publicly funded consortium has proceeded in three phases: genetic (linkage) mapping, physical mapping, and DNA sequencing.

Entire genomes can be mapped at the DNA level

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Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 20.12

Page 20: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• J. Craig Venter decided in 1992 to try a whole-genome shotgun approach.– This uses powerful computers to assemble

sequences from random fragments, skipping the first two steps.

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Genome sequences provide clues to important biological questions

Page 22: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• DNA microarray assays- detect and measure the expression of thousands of genes at one time– Tiny amounts of a large number of single-stranded

DNA fragments representing different genes are fixed on a glass slide in a tightly spaced array (grid).

– The fragments are tested for hybridization with various samples of fluorescently-labeled cDNA molecules.

Page 23: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same
Page 24: CHAPTER 20 DNA TECHNOLOGY AND GENOMICS. Recombinant DNA- genes from two different sources - often different species - are combined in vitro into the same

• Spots where any of the cDNA hybridizes fluoresce with an intensity indicating the relative amount of the mRNA that was in the tissue.

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• Proteomics, the systematic study of full protein sets (proteomes) encoded by genomes.

• Bioinformatics, the application of computer science and mathematics to genetic and other biological information

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• Gene Therapy

• Ethical issues in tinkering with our genome.

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– Transgenic organisms with genes from another species have been developed to exploit the attributes of the new genes (for example, faster growth, larger muscles).

– Other transgenic organisms are pharmaceutical “factories” - a producer of large amounts of an otherwise rare substance for medical use.

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• Scientists are using gene transfer to improve the nutritional value of crop plants.– For example, a transgenic rice plant has been

developed that produces yellow grains containing beta-carotene.• Humans use beta-carotene to make vitamin A.• Currently, 70% of children

under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.


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