Chapter DNA Tools and Biotechnology

Chapter 20 247 DNA Tools and Biotechnology
The recombinant DNA technology is a set of techniques used to combine the DNAs from different species of organisms in vitro, and to introduce this recombinant DNA (rDNA) into a host cell to produce biological products.

247 Restriction endonucleases (restriction enzymes) cut the phosphodiester bonds of both DNA strands at a specific sequence. The enzymes read the DNA sequence from 5’ to 3’ direction. Some enzymes cut the DNA strands in a staggered manner, resulting in the formation of DNA fragments with sticky ends.

Restriction enzyme cuts sugar-phosphate backbones.
Fig Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA

Fig Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA One possible combination

Fig Restriction site DNA 5 3 3 5 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule

248 The cell that produces a specific restriction endonuclease does not cut its own DNA because the bases within the sequences are protected from enzymatic action by methylation (methyl group, CH3, are added to the bases).

248 Cloning vectors: Plasmids: Bacteriophages:
Foreign genes can be spliced into a plasmid or a phage by using restriction enzymes and DNA ligase. The vectors are introduced into bacterial cells which are then cloned. The cells can synthesize the proteins encoded by the foreign genes.

248 Cloning Hosts: Bacteria: Escherichia coli Bacillus subtilis Yeasts

247 Gene-cloning procedure:
Isolation of DNA that contains the desired gene. Isolation of vector (plasmid) from bacteria. The plasmid carries two genes: ampR (resistant to ampicillin) and lacZ gene which codes for galactosidase, the enzyme that digests lactose. Cut both DNA and the plasmid with the same restriction endonuclease. The enzyme cuts the DNA into several thousand fragments. The enzyme also cuts the lacZ gene in the plasmid. The gene is disrupted and the cell is no longer able to digest the sugar (X-gal).

Cell containing gene of interest Bacterium
Fig. 20-2a Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome 2 2 Plasmid put into bacterial cell Figure 20.2 A preview of gene cloning and some uses of cloned genes Recombinant bacterium

Recombinant bacterium
Fig. 20-2b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested 4 Basic research and various applications Basic research on gene Basic research on protein Figure 20.2 A preview of gene cloning and some uses of cloned genes Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

4. Mix the fragments of DNA and the clipped plasmid.
5. Use DNA ligase to join the gene of interest and the plasmid to form a recombinant DNA (rDNA). 6. Introduce the rDNA into the bacterial cells by transformation

Grow the cells in a medium containing ampicillin and X-gal (a modified sugar).
8. The bacteria that carry recombinant plasmid can be identified by their resistance to ampicillin and their inability to digest X-gal. So, the colonies that can grow in the medium and are white in color are the ones that have picked up the foreign genes. The bacterial colonies that appear blue in color are the ones that do not have an insert and are able to produce the enzyme which digests the X-gal.

Identifying clones carrying a gene of interest:
a. Bacterial colonies are pressed against a special filter paper. b. The paper is treated with enzyme to break open the cells. c. The DNA is denatured by heat (93- 98oC) or alkaline solution such as NaOH to form single-stranded DNA on the paper.

d. The paper is incubated in a solution of the labeled probe.
e. The probe hybridizes with complementary DNA on the paper. f. The paper is rinsed and placed on a photographic film to allow exposure of the film to radioactive isotope. This technique is known as autoradiography. g. The developed film, an autoradiograph, is compared with the master plate to find out which colonies carry the desired gene.

247 The cells of the colony with the desired
gene are cloned in large quantities to produce the gene product. Genomic library: It is a set of DNA fragments from a genome, each is carried by a plasmid or a phage.

Fig. 20-5 Foreign genome cut up with restriction enzyme Large insert with many genes Large plasmid or BAC clone Recombinant phage DNA Bacterial clones Recombinant plasmids Phage clones Figure 20.5 Genomic libraries (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Foreign genome cut up with restriction enzyme
Fig. 20-5a Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial clones Recombinant plasmids Phage clones Figure 20.5a, b Genomic libraries (a) Plasmid library (b) Phage library

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

DNA in nucleus mRNAs in cytoplasm Fig. 20-6-1
Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene

Reverse transcriptase Poly-A tail mRNA
Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene DNA polymerase

Fig DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene DNA polymerase cDNA

248 Polymerase Chain Reaction (PCR): 1. Denaturation:
DNA to be amplified is denatured by heat at 95oC for 2 minutes. 2. Priming: DNA primers are added at 50-65oC for 2 minutes. The primers attach to the 3’ end of each denatured DNA strand. 3. Extension: DNA polymerase binds to the primer and extends the DNA from 5’ to 3’ direction. The extension is allowed to proceed for 2 minutes at 72oC.

molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8 TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Figure 20.8 The polymerase chain reaction (PCR) Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a
Figure 20.8 The polymerase chain reaction (PCR)

Cycle 1 yields 2 molecules
Fig. 20-8b 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension Figure 20.8 The polymerase chain reaction (PCR) New nucleotides

Cycle 2 yields 4 molecules
Fig. 20-8c Cycle 2 yields 4 molecules Figure 20.8 The polymerase chain reaction (PCR)

molecules; 2 molecules (in white boxes) match target sequence
Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Figure 20.8 The polymerase chain reaction (PCR)

248 Gel Electrophoresis: It is a technique of separating macromolecules such as nucleic acids or proteins on the basis of molecular size, electrical charge and other physical properties. Agarose gel: Cathode: Anode:

Figure 20.9 Gel electrophoresis
TECHNIQUE Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 Shorter molecules RESULTS Figure 20.9 Gel electrophoresis

Mixture of DNA molecules of different sizes
Fig. 20-9a TECHNIQUE Power source Mixture of DNA molecules of different sizes – Cathode Anode + Gel 1 Power source Figure 20.9 Gel electrophoresis – + Longer molecules 2 Shorter molecules

Fig. 20-9b RESULTS Figure 20.9 Gel electrophoresis

Fig Normal -globin allele Normal allele Sickle-cell allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 201 bp 175 bp 376 bp Large fragment DdeI Figure Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

248 Southern Blot: a technique that detects a specific DNA sequence by using a labeled probe which can be a DNA or a RNA.

Fig TECHNIQUE Heavy weight Restriction fragments DNA + restriction enzyme I II III Nitrocellulose membrane (blot) Gel Sponge I Normal -globin allele II Sickle-cell allele III Heterozygote Paper towels Alkaline solution 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting) Radioactively labeled probe for -globin gene Figure Southern blotting of DNA fragments Probe base-pairs with fragments I II III I II III Fragment from sickle-cell -globin allele Film over blot Fragment from normal -globin allele Nitrocellulose blot 4 Hybridization with radioactive probe 5 Probe detection

Restriction fragments DNA + restriction enzyme I II III
Fig a TECHNIQUE Heavy weight Restriction fragments DNA + restriction enzyme I II III Nitrocellulose membrane (blot) Gel Sponge I Normal -globin allele II Sickle-cell allele III Heterozygote Paper towels Alkaline solution Figure Southern blotting of DNA fragments 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled probe for -globin gene
Fig b Radioactively labeled probe for -globin gene Probe base-pairs with fragments I II III I II III Fragment from sickle-cell -globin allele Film over blot Figure Southern blotting of DNA fragments Fragment from normal -globin allele Nitrocellulose blot 4 Hybridization with radioactive probe 5 Probe detection

249 To facilitate the uptake of rDNA: Electrophoration:
Microscopic needles DNA (gene) gun

249 Northern Blot: a technique that detects a specific RNA sequence by using a labeled probe which can be a DNA or a RNA.

– Then we would synthesize this probe
A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5

Radioactively labeled probe molecules
Fig. 20-7 TECHNIQUE Radioactively labeled probe molecules Probe DNA Gene of interest Multiwell plates holding library clones Single-stranded DNA from cell Film • Figure 20.7 Detecting a specific DNA sequence by hybridizing with a nucleic acid probe Nylon membrane Nylon membrane Location of DNA with the complementary sequence

250 DNA Chip (DNA microarray assay):
Tiny amounts of a large number of single-stranded DNA fragments representing different genes are prepared and affixed (glued) to a glass slide. From the tissue sample, mRNA molecules are isolated. Single-stranded cDNA molecules are then synthesized from these mRNA molecules by using reverse transcriptase.

Labeled cDNA molecules (single strands)
Fig TECHNIQUE Tissue sample 1 Isolate mRNA. 2 Make cDNA by reverse transcription, using fluorescently labeled nucleotides. mRNA molecules Labeled cDNA molecules (single strands) 3 Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. DNA fragments representing specific genes Figure DNA microarray assay of gene expression levels DNA microarray DNA microarray with 2,400 human genes 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample.

250 These cDNA molecules are labeled with fluorescent dyes.
The labeled cDNAs are allowed to hybridize with the single-stranded DNAs on the DNA chip. The DNA chip is rinsed to remove the unbound labeled cDNAs. The DNA chip is viewed with a microscope which is attached to a computer. When a laser beam strikes the spots where hybridization occurs, they will fluoresce with an intensity indicating the relative amount of the mRNA that was in tissue.

251 Cloning Plants: Single-Cell Cultures
One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism

EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments
Fig EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments Figure Can a differentiated plant cell develop into a whole plant? Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. A single somatic carrot cell developed into a mature carrot plant.

252 Cloning Animals: Nuclear Transplantation
In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

EXPERIMENT RESULTS Frog embryo Frog egg cell Frog tadpole UV
Fig Frog embryo Frog egg cell Frog tadpole EXPERIMENT UV Fully differentiated (intestinal) cell Less differentiated cell Donor nucleus transplanted Donor nucleus transplanted Enucleated egg cell Egg with donor nucleus activated to begin development RESULTS Figure Can the nucleus from a differentiated animal cell direct development of an organism? Most develop into tadpoles Most stop developing before tadpole stage

252 Reproductive Cloning of Mammals
In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

TECHNIQUE RESULTS Mammary cell donor Egg cell donor
Fig TECHNIQUE Mammary cell donor Egg cell donor 1 2 Egg cell from ovary Nucleus removed Cultured mammary cells 3 Cells fused 3 Nucleus from mammary cell 4 Grown in culture Early embryo Figure Reproductive cloning of a mammal by nuclear transplantation For the Discovery Video Cloning, go to Animation and Video Files. 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor RESULTS

Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

Fig Figure CC, the first cloned cat, and her single parent

252 Problems Associated with Animal Cloning
In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

252 Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types The adult body also has stem cells, which replace nonreproducing specialized cells

From bone marrow in this example
Fig Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Figure Working with stem cells Different types of differentiated cells Liver cells Nerve cells Blood cells

The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

253 Medical Applications One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

Proteomics is the systemic study of the full protein sets encoded by genomes.
Bioinformatics is the application of computer science and mathematics to genetic and other biological information. Gene therapy is the treatment of genetic defects by introducing normal genes into the patients.

253 Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

Insert RNA version of normal allele into retrovirus.
Fig Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Figure Gene therapy using a retroviral vector Bone marrow 4 Inject engineered cells into patient.

Synthesis of Small Molecules for Use as Drugs
254 Synthesis of Small Molecules for Use as Drugs The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor Pharmaceutical products that are proteins can be synthesized on a large scale

254 Protein Production in Cell Cultures
Host cells in culture can be engineered to secrete a protein as it is made This is useful for the production of insulin, human growth hormones, and vaccines

Protein Production by “Pharm” Animals and Plants
Transgenic animals are made by introducing genes from one species into the genome of another animal Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use “Pharm” plants are also being developed to make human proteins for medical use

Fig Figure Goats as “pharm” animals

Forensic Evidence and Genetic Profiles
An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains Genetic profiles can be analyzed using RFLP analysis by Southern blotting

Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths The probability that two people who are not identical twins have the same STR markers is exceptionally small

Environmental Cleanup
Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

Agricultural Applications
DNA technology is being used to improve agricultural productivity and food quality

Animal Husbandry Genetic engineering of transgenic animals speeds up the selective breeding process Beneficial genes can be transferred between varieties or species

Genetic Engineering in Plants
Agricultural scientists have endowed a number of crop plants with genes for desirable traits The Ti plasmid is the most commonly used vector for introducing new genes into plant cells Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops

Agrobacterium tumefaciens
Fig TECHNIQUE Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA RESULTS DNA with the gene of interest Figure Using the Ti plasmid to produce transgenic plants For the Cell Biology Video Pronuclear Injection, go to Animation and Video Files. For the Discovery Video Transgenics, go to Animation and Video Files. Recombinant Ti plasmid Plant with new trait

Safety and Ethical Questions Raised by DNA Technology
Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology

Most public concern about possible hazards centers on genetically modified (GM) organisms used as food Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives

As biotechnology continues to change, so does its use in agriculture, industry, and medicine
National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology

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