11: Amplifying and Manipulating DNA Fragments

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11.1: Prelude to Molecular Genetics
Today, classical genetics is often complemented by molecular biology, to give molecular genetics, which involves the study of DNA and other macromolecules that have been isolated from an organism. Usually, molecular genetics experiments involve some combination of techniques to isolate and analyze the DNA or RNA transcribed from a particular gene.
11.2: Isolating Genomic DNA
DNA purification strategies rely on the chemical properties of DNA that distinguish it from other molecules in the cell, namely that it is a very long, negatively charged molecule. To extract purified DNA from a tissue sample, cells are broken open by grinding or lysing in a solution that contains chemicals that protect the DNA while disrupting other components of the cell (Figure 8.2). These chemicals may include detergents, which dissolve lipid membranes and denature proteins.
11.3: Isolating or Detecting a Specific Sequence by PCR
The Polymerase Chain Reaction (PCR) is a method of DNA replication that is performed in a test tube (i.e. in vitro). Here “polymerase” refers to a DNA polymerase enzyme extracted and purified from bacteria, and “chain reaction” refers to the ability of this technique produce millions of copies of a DNA molecule, by using each newly replicated double helix as a template to synthesize two new DNA double helices. PCR is therefore a very efficient method of amplifying DNA.
11.4: PCR and Gel Electrophoresis
The polymerase chain reaction laboratory technique is used in a variety of applications to make copies of a specific DNA sequence. This lesson describes how a PCR reaction works, what it accomplishes, and its basic requirements for success. Examples of interpreting results are given. PCR’s strengths, weaknesses, and applications to plant biotechnology are explained.
11.5: Origins of Molecular Polymorphisms
Some of mutations occur during DNA replication processes, resulting in an insertion, deletion, or substitution of one or a few nucleotides. Larger mutations can be caused by mobile genetic elements such as transposons, which are inserted more or less randomly into chromosomal DNA, sometimes occurring in clusters.
11.6: Classification and Detection of Molecular Markers
Mutations that do not affect the function of protein sequences or gene expression are likely to persist in a population as polymorphisms, since there will be no selection either in favor or against them (i.e. they are neutral). Note that the although the rate of spontaneous mutation in natural populations is sufficiently high so as to generate millions of polymorphisms that accumulate over thousands of generations, the rate of mutation is slow.
11.7: Cutting and Pasting DNA- Restriction Digests and DNA Ligation
Many bacteria have enzymes that recognize specific DNA sequences and then cut the double stranded DNA helix at this sequence. These enzymes are called site-specific restriction endonucleases, or more simply “restriction enzymes”, and they naturally function as part of bacterial defenses against viruses and other sources of foreign DNA. To cut DNA at known locations, researchers use restriction enzymes from various bacterial species, and which can be purchased from various commercial sources.
11.8: 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.
11.9: 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.
11.10: 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.
11.11: 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!