Chapter 20: Biotechnology

Chapter 20: Biotechnology

20.1 and 20.2 are the only sections we will cover in Chapter 20. The rest will be covered after the AP exam. • Self Quiz Multiple Choice Questions p. 424 – 425 (1-8) and question 9

Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • Nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

Genetic Engineering • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

20.1 DNA cloning yields multiple copies of a gene or other DNA segment

Cloning • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning

Use of bacteria in cloning • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids – mainly E. coli • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome • Plasmid DNA may not always be needed for cell processes • Cloned genes are useful for making copies of a particular gene and producing a protein product

Gene Cloning Using Bacteria • Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid • The recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene

Fig. 20-2a Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium

Fig. 20-2b Recombinantbacterium Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest 3 Protein expressedby gene of interest Gene ofInterest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste

Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments • DNA ligase

Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end

Fig. 20-3-2 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination

Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • DNA molecule that can carry foreign DNA into a host cell and replicate there • Bacterial plasmids are useful to clone eukaryotic DNA: • Easy to isolate, manipulate and reintroduce • Multiply rapidly

Hummingbird β-globin Gene Cloning Turn to page 399. Summarize the steps in hummingbird β-globin gene cloning.

Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments

Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids

Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

Hummingbird β-globin Gene Cloning Follow Up Questions • What two genes were targeted in the hummingbird cloning experiment. • Where was the DNA cut? What enzyme was used to cut the DNA? • What enzyme was responsible for covalently bonding the “sticky ends” • Do all cells take up recombinant plasmids? • What was included in the nutrient agar? • Describe the experimental results.

Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome • Plasmid clones contain copies of a particular gene within a plasmid • A genomic library that is made using bacteriophages is stored as a collection of phage clones • Normal infection process will produce many copies of the cloned DNA

Fig. 20-5 Foreign genomecut up withrestrictionenzyme Large insertwith many genes Large plasmid or BACclone Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Fig. 20-5a Foreign genomecut up withrestrictionenzyme or Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library

BACs • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert • BACs are another type of vector used in DNA library construction

cDNA • A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization

Expressing Cloned Eukaryotic Genes • After a gene has been cloned, its protein product can be produced in larger amounts for research • Cloned genes can be expressed as protein in either bacterial or eukaryotic cells

Bacterial Expression Systems • Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells • Differences in promoter/control sequences • Presence of introns • To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter • Use of cDNA can overcome issues with introns

Eukaryotic Cloning and Expression Systems • To avoid gene expression problems • Use of cultured eukaryotic cells as host cells • Yeasts and single-celled fungi – easy to grow, contain plasmids • Yeast artificial chromosomes (YACs) • Behave like normal chromosomes in mitosis • Can carry more DNA than a plasmid • Eukaryotic hosts can provide the post-translational modifications that many proteins require

Eukaryotic Cloning and Expression Systems • One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes • Alternatively, scientists can inject DNA into cells using microscopically thin needles • Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

How does PCR work? • Heat denatures the DNA • Cooling allows hydrogen bonds to reform between the strands of DNA • Heat-stable DNA polymerase base pairing from target segment of DNA

Fig. 20-8a 5 3 TECHNIQUE Targetsequence Genomic DNA 3 5

Fig. 20-8b 1 5 3 Denaturation 3 5 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides

Fig. 20-8c Cycle 2yields 4 molecules

Fig. 20-8d Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

Quick Review • Define and distinguish between genomic libraries using plasmids, phages, and cDNA • Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure

Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene

Gel Electrophoresis • Pg. 405 • Fragments of DNA (obtained through restriction enzyme digestion) or PCR amplification are separated into bands based on physical differences • Size • Electrical charge • Other physical properties • Utilizes gel made of a polymer (usually polysaccharide) and an electrical field.

The gel is made using agarose (similar to gelatin, but made of seaweed), a buffer (salt water solution to allow electrical charges to flow through the gel), a flask, a microwave, the gel mold, and a gel comb. The agarose and buffer are mixed, microwaved, and poured into the gel mold. • The comb is placed in one end of the gel (to make the wells) and the gel cools. • To set up the electrophoresis box, buffer is poured into the box and the gel (still in the mold) is also placed in the box. • A loading buffer (to dye the DNA) is added to the DNA tube and the DNA samples are placed in each of the wells at the top of the gel using a pippetor, taking care not to break the gel. These wells can be seen as the darker rectangles at the top of the picture to the right.

Use a pippetor to place DNA size standard in the well next to the first well you placed the DNA in. This is used as a "ruler" to measure the DNA bands later. • An electric field is set up across the gel with the negative (-) end at the top where the wells are and the positive (+) end opposite the wells. When an electrical current is added the DNA will move towards the positive charge because DNA has a negative charge. • The short strands will move faster (and therefore farther) than the longer strands (due to some basic laws of physics) and strands of the same length will move the same distance. Thus, the DNA arranges itself on the gel according to length, longest strands being nearest to the starting wells. • The sorted DNA on the gel is stained with ethidium bromide so that it becomes visible when UV light is shown on the gel. The photo to the right represents a gel electrophoresis. You are not seeing individual strands of DNA, but rather groups of DNA of the same length (represented as the white bands). • The DNA strand lengths are measured in base pairs (bp) based on the DNA size standard bands.

Work with a partner to summarize two of the DNA techniques. • You will present your summary to the class.

Gene Sequence Determination • Gel electrophoresis • Restriction fragment analysis • Southern blotting • Dideoxyribonucleotide chain termination method

Gene Expression Determination • Northern blotting • Reverse transcriptase-polymerase chain reaction • In situ hybridization • DNA microarray assays

Gene Function Determination • In vitro mutagenesis • RNA interference (covered in chapter 18)

Quick Review • Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene • Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR

Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications

Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

Chapter 20: Biotechnology