recap

18.3 recap

DNA for cloning can be obtained from genomic libraries, cDNA, or artificially synthesized DNA fragments.

learning outcomes

You should be able to:

  • Determine which type of library would be most appropriate to construct for a given scenario.

  • Predict the expected results in terms of quantities and sequences of cDNA libraries taken from different tissues.

  • Show how RT-PCR is used to amplify a specific gene sequence.

  • Summarize uses of synthetic DNA.

Question 1

What type of gene library would you use for analyzing each of the following, and why?

  1. The expression of a gene encoding amylase, a starch hydrolyzing enzyme, in seeds as they germinate

  2. The sequence of the amylase gene in two different genetic strains of corn

  1. This involves tissue-specific gene expression, so use RT-PCR, which is a “snapshot” of the mRNAs in the cell at a given time and can identify the amylase mRNA.

  2. This calls for genomic analysis, so use a genomic library and identity the amylase gene in the strains.

Question 2

How could RT-PCR be used to compare the mRNA’s made by muscle vs. liver tissues?

All cellular RNA is isolated and cDNA copies of the RNAs are made using reverse transcriptase. Primers for DNA replication are made for the two ends of the DNA for the specific gene whose expression is to be studied. Then a PCR procedure is carried out using these primers. The degree of amplification from these primers is proportional to the specific cDNA target, which in turn is proportional to the amount of the specific RNA in the original cell extract.

Question 3

Describe a use for synthetic DNA.

Synthetic DNA can be used to make a probe for hybridization to identify a genotype, as in genetic screening for human diseases. Mutations in the DNA can be used to detect mutations in patients.

Question 4

Explain why a cDNA library constructed from human brain tissue will likely be both similar to and different from a cDNA library constructed from human pancreatic tissue.

Both tissues express some identical genes that are involved in cellular activities, such as energy metabolism (e.g., genes encoding enzymes in the citric acid cycle) and informational functions (e.g., making rRNA). The tissues will differ in tissue-specific mRNAs. For example, the brain makes proteins needed for electrical activity and communication between cells, while the pancreas makes the hormone insulin. But note that all tissues have the same DNA sequences—all of them.

388

We’ve explored the various sources of DNA that can be used to make recombinant DNA molecules, and how organisms are transformed with recombinant DNA. We will now turn to some of the ways that recombinant DNA and transformation methods can be used to study the functions of genes and proteins.