Chapter 19: Recombinant DNA techniques --- Comprehension Questions

Here, 4n = 1,048,500. So n = 10; a 10-bp recognition sequence is most likely. 10

A microbiologist discovers a new type II restriction endonuclease. When DNA is digested by this enzyme, fragments that average 1,048,500 bp in length are produced. What is the most likely number of base pairs in the recognition sequence of this enzyme?

By staining with a fluorescent dye such as ethidium bromide, which intercalates between the stacked bases of the DNA double helix.

After DNA fragments have been separated by gel electrophoresis, how can they be visualized?

1) Restriction cloning: Vector and foreign DNA are cut with the same restriction enzyme, then ligated together with DNA ligase. This is the most straightforward, simplest method of cloning. The disadvantage is that matching restriction sites may not be available. Ligation also produces undesirable products, such as the vector ligating to itself, without the foreign DNA insert. 2) Ligation of blunt ends with T4 DNA ligase: Any two blunt ends of DNA fragments can be ligated, so the method does not depend on the presence of suitable restriction sites. The disadvantage is that blunt-end ligations are inefficient, and vector self-ligation is difficult to control.

Briefly describe two different methods for inserting foreign DNA into plasmids, giving the strengths and weaknesses of each method.

Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids and transform the plasmids into E. coli cells. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid, an inducer of the lac operon, and X-gal, a substrate for β-galactosidase that turns blue when cleaved. Colonies that carry the plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry the plasmid with foreign DNA inserts will not make functional β-galactosidase and will remain white.

Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid.

First, the double-stranded template DNA is denatured by high temperature. Then, synthetic oligonucleotide primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension doubles the number of copies of the target sequence between the primers. A limitation of PCR amplification is that, to synthesize the PCR primers, the sequence of the gene to be amplifi ed must be known, at least at the ends of the region to be amplified. Another is that the extreme sensitivity of the technique renders it susceptible to contamination. A third limitation is that the most-common thermostable DNA polymerase used for PCR, Taq DNA polymerase, has a high error rate. A fourth limitation is that PCR amplifi cation is usually limited to DNA fragments of a few thousand base pairs or less; optimal DNA polymerase mixtures and reaction conditions extend the amplifi able length to about 20 kb.

Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence. What are some of the limitations of PCR?

Gel electrophoresis uses an electric fi eld to drive negatively charged DNA molecules through a gel that acts as a molecular sieve. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules.

Explain how gel electrophoresis is used to separate DNA fragments of different lengths.

Cloning vectors should have (i) an origin of DNA replication so they can be maintained in a cell; (ii) a gene, such as an antibioticresistance gene, to select for cells that carry the vector; and (iii) a unique restriction site or series of sites at which a foreign DNA molecule can be inserted.

Give three important characteristics of cloning vectors.

The recognition sequences are palindromic and 4-8 base pairs long.

What common feature is seen in the sequences recognized by type II restriction enzymes?

DNA fingerprinting detects genetic differences among people by using probes for highly variable regions (usually, microsatellites, or short tandem repeats) of chromosomes. Typically, PCR is used to detect the microsatellites. Because people differ in the number of repeated sequences that they possess, the technique is used in the analysis of crimes, in paternity cases, and in identifying remains.

What is DNA fingerprinting? What types of sequences are examined in DNA fingerprinting?

Southern blotting is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe. DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. The membrane filter now has single-stranded DNA fragments bound to its surface, separated by size as in the gel. The filter is then incubated with a solution containing a denatured, labeled probe DNA. The probe DNA hybridizes to its complementary DNA on the filter. After washing excess unbound probes, the labeled probe hybridized to the DNA on the filter can be detected using the appropriate methods to visualize the label. For radioactively labeled probes, the bound probe is detected by exposure to X-ray film.

What is the purpose of Southern blotting? How is it carried out?

incorporation of a ddNTP (which lacks a 3' OH) into a strand of DNA prevents additional polymerization on that strand

What is the purpose of the dideoxynucleoside triphosphate in the dideoxy sequencing reaction?

Restriction enzymes cut foreign DNA, such as viral DNA, into fragments. Bacteria protect their own DNA by modifying bases, usually by methylation, at the recognition sites.

What role do restriction enzymes play in bacteria? How do bacteria protect their own DNA from the action of restriction enzymes?