Chapter Summary, Questions Answers - Biotechnology and Human Disease

CHAPTER SUMMARY

Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA (dsDNA) into smaller fragments. Each enzyme cleaves at a specific four to eight–base pair sequence (a restriction site), producing DNA segments called restriction fragments. The sequences that are recognized are palindromic. Restriction enzymes form either staggered cuts (sticky ends) or blunt-end cuts on the DNA. Bacterial DNA ligases can join two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This hybrid combination of two fragments is called a recombinant DNA molecule. Introduction of a foreign DNA molecule into a replicating cell permits the amplification (production of many copies) of the DNA, a process called cloning. A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium (or artificially constructed); joined with the DNA of interest; and reintroduced into the bacterium, which will replicate, thus making multiple copies of the hybrid plasmid. A DNA library is a collection of cloned restriction fragments of the DNA of an organism. A genomic library is a collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of every DNA nucleotide sequence in the genome. In contrast, complementary DNA (cDNA) libraries contain only those DNA sequences that are complementary to messenger RNA (mRNA) molecules present in a cell and differ from one cell type to another. Because cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of human proteins by bacteria or eukaryotes. Cloned, then purified, fragments of DNA can be sequenced, for example, using the Sanger dideoxy method. A probe is a small piece of RNA or single-stranded DNA (usually labeled with a radioisotope, such as 32P, or another recognizable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA). Probes can be used to identify which clone of a library or which band on a gel contains the target DNA. Southern blotting is a technique that can be used to detect specific sequences present in DNA. The DNA is cleaved using a restriction endonuclease, and the pieces are separated by gel electrophoresis and are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. The human genome contains many thousands of polymorphisms (DNA sequence variations at a given locus). Polymorphisms can arise from single-base changes and from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution in one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site also can be created by the same mechanism. In either case, cleavage with the endonuclease results in fragments of lengths differing from the normal that can be detected by DNA hybridization. This technique can be used to diagnose genetic diseases early in the gestation of a fetus. The polymerase chain reaction (PCR), another method for amplifying a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source. Applications of the PCR technique include: 1) efficient comparison of a normal gene with a mutant form of the gene, 2) detection of low-abundance nucleic acid sequences, 3) forensic analysis of DNA samples, and 4) prenatal diagnosis and carrier detection (for example, of cystic fibrosis). The products of gene expression (mRNA and proteins) can be measured by techniques such as the following: Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene expression in two different types of cells (for example, normal and cancer cells); enzyme-linked immunosorbent assays a n d Western blots (immunoblots) are used to detect specific proteins. Proteomics is the study of all the proteins expressed by a genome. The goal of gene therapy is the insertion of a normal cloned gene to replace a defective gene in a somatic cell. Insertion of a foreign gene into the germline of an animal creates a transgenic animal that can produce therapeutic proteins or serve as a model for human diseases.

Study Questions
Choose the ONE best answer.

33.1 HindIII is a restriction endonuclease. Which of the following is most likely to be the recognition sequence for this enzyme?

A. AAGAAG

B. AAGAGA

C. AAGCTT

D. AAGGAA

E. AAGTTC

Correct answer = C. The vast majority of restriction endonucleases recognize palindromes in double-stranded DNA, and AAGCTT is the only palindrome among the choices. Because the sequence of only one DNA strand is given, the base sequence of the complementary strand must be determined. To be a palindrome, both strands must have the same sequence when read in the 5I →3I direction. Thus, the complement of 5I -AAGCTT-3 I is also 5I -AAGCTT-3 I.

33.2 An Ashkenazi Jewish couple brings their 6-month-old son to you for evaluation of listlessness, poor head control, and a fixed gaze. You determine that he has Tay-Sachs disease, an autosomal recessive disorder. The couple also has a daughter. The family’s pedigree is shown to the right, along with Southern blots of a restriction fragment length polymorphism very closely linked to the gene for hexosaminidase A, which is defective in Tay-Sachs. Which of the statements below is most accurate with respect to the daughter?

A. She has a 25% chance of having Tay-Sachs disease.

B. She has a 50% chance of having Tay-Sachs disease.

C. She has Tay-Sachs disease.

D. She is a carrier for Tay-Sachs disease.

E. She is homozygous normal.

Correct answer = E. Because they have an affected son, both the biological father and mother must be carriers for this disease. The affected son must have inherited a mutant allele from each parent. Because he shows only the 3-kilobase (kb) band on the Southern blot, the mutant allele for this disease must be linked to the 3-kb band. The normal allele must be linked to the 4-kb band, and, because the daughter inherited only the 4-kb band, she must be homozygous normal for the hexosaminidase A gene.

33.3 A physician would like to determine the global patterns of gene expression in two different types of tumor cells in order to develop the most appropriate form of chemotherapy for each patient. Which of the following techniques would be most appropriate for this purpose?

A. Enzyme-linked immunosorbent assay

B. Microarray

C. Northern blot

D. Southern blot

E. Western blot

Correct answer = B. Microarray analysis allows the determination of messenger RNA (mRNA) production (gene expression) from thousands of genes at once. A Northern blot only measures mRNA production from one gene at a time. Western blots and enzyme-linked immunosorbent assay measure protein production (also gene expression) but only from one gene at a time. Southern blots are used to analyze DNA, not DNA expression.

33.4 A 2-week-old infant is diagnosed with a urea cycle defect. Enzymic analysis showed no activity for ornithine transcarbamoylase (OTC), an enzyme of the cycle. Molecular analysis revealed that the messenger RNA (mRNA) product of the gene for OTC was identical to that of a control. Which of the techniques listed below was most likely used to analyze mRNA?

A. Dideoxy chain termination

B. Northern blot

C. Polymerase chain reaction

D. Southern blot

E. Western blot

Correct answer = B. Northern blot allows analysis of the messenger RNA present (expressed) in a particular cell or tissue. Southern blot is used for DNA analysis, whereas Western blot is used for protein analysis. Dideoxy chain termination is used to sequence DNA. Polymerase chain reaction is used to generate multiple, identical copies of a DNA sequence in vitro.

33.5 For the patient above, which phase of the central dogma was most likely affected?

Correct answer = Translation. The gene is present and is able to be expressed as evidenced by messenger RNA production. The lack of enzymic activity means that some aspect of protein synthesis is affected.