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9.3 Applications of DNA Sequencing

1. The nucleotide sequence of a gene can be used to determine the amino acid sequence of the protein for which it codes, identify genetic alterations that result in certain diseases, and study evolutionary relatedness.

9.1 Applications of Genetic Engineering (Table 9.2)

Genetically Engineered Bacteria (Figure 9.2)

1. Bacteria can be engineered to produce pharmaceutical proteins, vaccines, and other proteins more efficiently.

2. By cloning a segment of DNA into E. coli, an easy source of that sequence is available for study and further manipulation.

3. Gene function and regulation can more easily be studied in E. coli because systems for manipulating its DNA have been developed.

Genetically Engineered Eukaryotes

1. Transgenic plants can be made using a vector derived from the

Ti plasmid of Agrobacterium tumefaciens. Examples include plants that resist pests and herbicides, plants with improved nutritional value, and plants that function as edible vaccines.

9.2 Applications of Nucleic Acid Hybridization (Figure 9.6)

Colony Blots and Southern Blots

1. A probe can be used to locate a specific nucleotide sequence in a DNA sample.

2. Colony blots are used to identify colonies that contain a given sequence of DNA. (Figure 9.7)

3. Southern blots are used to determine the size of the restriction fragments that contain a sequence of interest and to detect subtle variations in nucleotide sequences that occur in different strains of a given species. (Figure 9.8)

Fluorescence in situ Hybridization (FISH)

1. Fluorescence in situ hybridization (FISH) uses a fluorescently-labeled probe to detect specific nucleotide sequences within intact cells affixed to a microscope slide.

Nucleotide Array Technologies

1. Nucleotide array technologies employ a microarray containing tens or hundreds of thousands of oligonucleotides; each oligonucleotide functions in a manner analogous to a probe. (Figure 9.10)

9.4 Applications of the Polymerase Chain Reaction

1. The polymerase chain reaction (PCR) is used to rapidly increase the amount of a specific DNA segment in a sample. (Figure 9.11)

9.5 Concerns Regarding DNA Technologies

1. Advances in genomics raise ethical issues and concerns about confidentiality.

2. Genetically modified organisms hold many promises, but concerns exist about the introduction of allergens into a food product and adverse effects on the environment.

Recombinant DNA Techniques

9.6 Techniques Used in Genetic Engineering

Genetically Engineering Bacteria

1. An approach frequently used to clone a specific bacterial gene is to first clone into a population of E. coli cells a set of DNA fragments that together make up the entire genome of the organism being studied, creating a DNA library. (Figure 9.12)

2. To isolate DNA, cells are lysed by adding a detergent. In order to obtain eukaryotic DNA without introns, reverse transcriptase is used to make a copy of DNA from an mRNA template. (Figure 9.13)

3. DNA is cut into smaller fragments by digesting it with a restriction enzyme. (Figure 9.14)

4. The enzyme DNA ligase is used to join the vector and the insert. A vector typically has an origin of replication, a selectable marker, and a second genetic marker that contains within it a multiple-cloning site. (Figures 9.15 and 9.16)

5. The type of vector used to clone eukaryotic DNA depends largely on the ultimate purpose of the procedure; they include expression vectors and bacterial artificial chromosomes.

6. For routine cloning experiments, a strain of E. coli is used as a host.

7. The recombinant molecule is introduced into the new host using transformation or electroporation.

8. The transformed cells are cultivated on medium that both selects for cells containing vector sequences and differentiates those that carry recombinant plasmids.

Genetically Engineering Eukaryotic Cells

1. Vectors used to transfer DNA into eukaryotic cells include the Ti plasmid, artificial chromosomes, and viruses.

2. Ti plasmids and viruses naturally carry DNA into eukaryotic cells. Electroporation and a gene gun can be used to move DNA into eukaryotic cells. (Figure 9.17)

9.7 Techniques Used in Nucleic Acid Hybridization

Techniques Used in Colony Blotting and Southern Blotting

1. A gene similar to the one being sought can be used as a probe, or an appropriate oligonucleotide can be synthesized.

2. To do a colony blot, colonies are replica-plated onto a nylon membrane; a DNA probe is then used to identify colonies that contain the sequence of interest.

3. A Southern blot involves several steps. Gel electrophoresis is used to separate DNA fragments according to size, the separated DNA is transferred in-place to a nylon membrane, and then DNA probe is added to the membrane to locate specific nucleotide sequences. (Figure 9.18)

Techniques Used in Fluorescence in situ Hybridization

1. Samples must be treated to preserve the shape of the cells, inactivate enzymes, and make the cells permeable.

Techniques Used in Nucleotide Array Technologies

1. Sophisticated techniques are used to construct microarrays. The DNA to be analyzed is labeled and added to the microarray.

Review Questions

9.8 Techniques Used in DNA Sequencing

Dideoxy Chain Termination Method

1. A key ingredient in a sequencing reaction is a dideoxynucleotide, a nucleotide that lacks the 3'OH and therefore functions as a chain terminator.

2. The sizes of fragments in a sequencing reaction indicate the positions of the terminating nucleotide base in the synthesized DNA strand. (Figures 9.20 and 9.21)

Automated DNA Sequencing

1. Each different ddNTP is labeled with a different color of fluorescent dye; the reactions can be done in one tube and run on one lane of a gel; a laser detects the color of the band as it runs off the gel. (Figure 9.22)

9.9 Techniques Used in the Polymerase Chain Reaction (PCR)

The Three-Step Amplification Cycle (Figure 9.23)

1. Double-stranded DNA is denatured, primers anneal to their complementary sequences, and then DNA is synthesized, amplifying the target sequence.

Generating a Discrete-Sized Fragment (Figure 9.24)

1. A discrete-sized fragment that is amplified exponentially is obtained after three cycles of replication; the size of the amplified fragment is dictated by the positions to which the primers annealed.

Selecting Primer Pairs

1. The two primers that are selected dictate which portion of the DNA is amplified.

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