Essential concepts

Question 1

Trangenesis techniques

Answer 1

Developed for well-studied eukaryotic species.
Depend on an understanding of the reproductive biology of the species.
Potential to serve important roles in agriculture, biotechnology & human gene therapy.

Question 2

Targeted gene knockouts (KO)

Answer 2

Deletion or disruption of a specific gene/protein to study the resultant phenotype.
Methods for yeast, mouse, drosophilia.
KO methods using zinc-finger nuclease/TALEN/CRISPR-Cas9 can be universally applied to various species.

Question 3

How do forward genetic mutagenesis screening begin?

Answer 3

1. Isolation of mutants that show phenotype of interest.
2. After cloning of the gene you can deduce the function of the gene in the specific physiological process.

Question 4

What does positional cloning identify?

Answer 4

The genes responsible for traits whose molecular cause is unknown.

Question 5

How to localize a trait-affecting gene to specific region of chromosal DNA?

Answer 5

Combine formal linkage analysis with the use of DNA markers.

Question 6

What revolutionized the cloning of mutant genes?

Answer 6

SNP (single nucleotide polymorphism) with known genome sequences of model organisms.

Question 7

What does confirmation of gene require?

Answer 7

Phenotypic rescue by wild-type transgene.

Question 8

Recombinant DNA technology

Answer 8

1.Cut enormously long strings of DNA into much smaller fragments with restriction enzymes and separate them by gel electrophoresis.
2.
Isolate, amplify, and purify fragments through molecular cloning
.
3. Use purified DNA fragments as hybridization probes to identify similar sequences in libraries of clones or mixtures of DNA or RNA molecules
.
4. Rapidly isolate and amplify previously defined genomic or mRNA sequences through the polymerase chain reaction (PCR)
.
5.Determine the precise sequence of bases within isolated DNA fragments