Deck 18: Molecular Genetic Analysis and Biotechnology

Question 1

Type II restriction enzymes cut DNA at specific base sequences that are palindromic. Some restriction enzymes make staggered cuts, producing DNA fragments with cohesive ends; others cut both strands straight across, producing blunt-ended fragments. There are fewer long recognition sequences in DNA than short sequences.
-Where do restriction enzymes come from?

Answer 1

Restriction enzymes exist naturally in bacteria, which use them to prevent the entry of viral DNA.

Question 2

DNA fragments can be separated, and their sizes can be determined with the use of gel electrophoresis. The fragments can be viewed by using a dye that is specific for nucleic acids or by labeling the fragments with a radioactive or chemical tag.

-DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel?

A) 2000-bp fragment
B) 1000-bp fragment
C) 500-bp fragment
D) All will migrate equal distances.

Answer 2

500-bp fragment

Question 3

Labeled probes, which are sequences of RNA or DNA that are complementary to the sequence of interest, can be used to locate individual genes or DNA sequences. Southern blotting can be used to transfer DNA fragments from a gel to a membrane such as nitrocellulose.
-How do Northern and Western blotting differ from Southern blotting?

Answer 3

Southern blotting is used to transfer DNA from a gel to a solid medium. Northern blotting is used to transfer RNA from a gel to a solid medium, and Western blotting is used to transfer protein from a gel to a solid medium.

Question 4

DNA fragments can be inserted into cloning vectors, stable pieces of DNA that will replicate within a cell. A cloning vector must have an origin of replication, one or more unique restriction sites, and selectable markers. An expression vector contains sequences that allow a cloned gene to be transcribed and translated. Special cloning vectors have been developed for introducing genes into eukaryotic cells.
-How is a gene inserted into a plasmid cloning vector?

Question 5

The polymerase chain reaction is an enzymatic in vitro method for rapidly amplifying DNA. In this process, DNA is heated to separate the two strands, short primers attach to the target DNA, and DNA polymerase synthesizes new DNA strands from the primers. Each cycle of PCR doubles the amount of DNA. PCR has a number of important applications in molecular biology.
-Why is the use of a heat-stable DNA polymerase important to the success of PCR?

Question 6

A DNA library can be screened for a specific gene with the use of complementary probes that hybridize to the gene. Alternatively, the library can be cloned into an expression vector, and the gene can be located by examining the clones for the protein product of the gene.
-Briefly explain how synthetic probes are created to screen a DNA library when the protein encoded by the gene is known.

Question 7

Positional cloning allows researchers to isolate a gene without having knowledge of its biochemical basis. Linkage studies are used to map the locus producing a phenotype of interest to a particular chromosome region. Chromosome walking and jumping are used to progress from molecular makers to clones containing sequences that cover the chromosome region. Candidate genes within the region are then evaluated to determine if they encode the phenotype of interest.
-How are candidate genes that are identified by positional cloning evaluated to determine whether they encode the phenotype of interest?

Question 8

The dideoxy sequencing method uses ddNTPs, which terminate DNA synthesis at specific bases.

-In the dideoxy sequencing reaction, what terminates DNA synthesis at a particular base?

A) The absence of a base on the ddNTP halts the DNA polymerase.
B) The ddNTP causes a break in the sugar-phosphate backbone.
C) DNA polymerase will not incorporate a ddNTP into the growing DNA strand.
D) The absence of a 3'-OH group on the ddNTP prevents the addition of another nucleotide

Question 9

DNA fingerprinting detects genetic differences among people by using probes for highly variable regions of chromosomes.
-How are microsatellites detected?

Question 10

Forward genetics begins with a phenotype and detects and analyzes the genotype that causes the phenotype. Reverse genetics begins with a gene sequence and, through analysis, determines the phenotype that it encodes. Particular mutations can be introduced at specific sites within a gene by means of site-directed and oligonucleotide-directed mutagenesis.

-A geneticist interested in immune function induces random mutations in a number of specific genes in mice and then determines which of the resulting mutant mice have impaired immune function. This procedure is an example of

A) forward genetics.
B) reverse genetics.
C) both forward and reverse genetics.
D) neither forward nor reverse genetics.

Question 11

A transgenic mouse is produced by the injection of cloned DNA into the pronucleus of a fertilized egg, followed by implantation of the egg into a female mouse. In knockout mice, the injected DNA contains a mutation that disables a gene. Inside the mouse embryo, the disabled copy of the gene can exchange with the normal copy of the gene through homologous recombination.

-What is the advantage of using the neo gene to disrupt the function of a gene in knockout mice?

A) The neo gene produces an antibiotic that kills unwanted cells.
B) The neo gene is the right size for disabling other genes.
C) The neo gene provides a selectable marker for finding cells that contain the disabled gene.
D) The neo gene produces a toxin that inhibits transcription of the target gene.

Question 12

Recombinant DNA technology is used to create a wide range of commercial products, including pharmaceutical products, specialized bacteria, genetically engineered crops, and transgenic domestic animals.
-What are some matters of concern about the use of genetically engineered crops?

Question 13

Gene therapy is the direct transfer of genes into humans to treat disease. Gene therapy was first successfully implemented in 1990 and is now being used to treat genetic diseases, cancer, and infectious diseases.
-What is the difference between somatic gene therapy and germ-line gene therapy?