15: DNA Technologies
Learning Objectives
- Suggest molecular techniques to design experiments (e.g., how would you use cDNA or a PCR product to clone a gene).
- Determine when to make or use a cDNA library or a genomic library
- Outline an experiment to purify rRNA from eukaryotic cells.
- Outline an experiment to isolate a cDNA for a human protein and clone it so you can manufacture insulin for the treatment of disease.
- Explain why you might want to clone and express a human growth hormone gene.
- List components needed to make a cDNA library using purified poly(A) RNA.
- List the components needed to make a genomic library from isolated genomic DNA.
- Compare PCR and genomic cloning as strategies for isolating a gene.
- Outline a strategy for using fly DNA to obtain copies of a human DNA sequence.
- Ask a question that requires screening a genomic library to obtain a gene you want to study
- Ask a question that requires using a microarray to obtain a gene you want to study
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- 15.1: Overview
- We start this chapter by looking at technologies that led to genetic engineering. The ability of make recombinant DNA is such a seminal technology that just realizing it could be done and then doing it in a test tube for the first time earned Paul berg a half-share in the 1980 Nobel Prize in Chemistry (the other half was shared by Walter Gilbert and Frederick Sanger for studies that enabled efficient DNA sequencing).
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- 15.2: Make and Screen a cDNA Library
- The first step in making a cDNA library is to isolate cellular mRNA. This mRNA extract should represent all of the transcripts in the cells at the time of isolation, or the cell’s transcriptome. This term is used by analogy to genome. However, a genome is all of the genetic information of an organism. In contrast, a transcriptome (usually eukaryotic) reflects all of the genes expressed in a given cell type at a moment in time.
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- 15.3: DNA Sequencing
- RNA sequencing came first, when Robert Holley sequenced a tRNA in 1965. The direct sequencing of tRNAs was possible because tRNAs are small, short nucleic acids, and because many of the bases in tRNAs are chemically modified after transcription. An early method for DNA sequencing developed by Walter Gilbert and colleagues involved DNA fragmentation, sequencing of the small fragments of DNA, and then aligning the overlapping sequences of the short fragments to assemble longer sequences.
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- 15.4: Genomic Libraries
- A genomic library might be a tube full of recombinant bacteriophage. Each phage DNA molecule contains a fragmentary insert of cellular DNA from a foreign organism. The library is made to contain a representation of all of possible fragments of that genome. The need for vectors like bacteriophage that can accommodate long inserts becomes obvious from the following bit of math.
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- 15.5: The Polymerase Chain Reaction (PCR)
- The polymerase chain reaction (PCR) can amplify a region of DNA from any source, even from a single cell’s worth of DNA or from fragments of DNA obtained from a fossil. This amplification usually takes just a few hours, generating millions of copies of the desired target DNA sequence. The effect is to purify the DNA from surrounding sequences in a single reaction!
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- 15.6: Genomic Approaches- The DNA Microarray
- Traditionally, when cellular levels of a protein were known to change in response to a chemical effector, molecular studies focused on control of the transcription of its gene. These studies often revealed that the control of gene expression was at the level of transcription, turning a gene on or off through interactions of transcription factors with DNA.
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- 15.7: Ome-Sweet Ome
- Early molecular technologies, including the ones described in this chapter, were applied to understanding the structure, function and regulation of specific genes. Some of the more recent technologies (e.g., microarrays) are well adapted to holistic approaches to understanding cell function. Terms we have already seen (genome, epigenome, transcriptome) were coined in an effort to define the different objects of study whose underlying network of molecular interactions can more accurately explain
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- 15.8: From Genetic Engineering and Genetic Modification
- By enabling us to focus on how genes and their regulation have evolved, these genomic, transcriptomic and proteomic technologies have vastly increased our knowledge of how cells work at a molecular level. We continue to add to our knowledge of disease process and in at least a few cases, how we can treat disease.