Deck 3: Recombinant Dna Technology and Genomics

Question 1

Distinguish among gene cloning, recombinant DNA technology, and genetic engineering by describing each process and discussing how they are interrelated. Provide examples of each approach as described in this chapter.

Answer 1

The terms recombinant DNA (deoxyribonucleic acid) technology, gene cloning, and genetic engineering are used to describe the same technique. These techniques differ slightly in their methodologies that are interrelated.
Gene cloning can be defined as the replication of fragments of deoxyribonucleic acid (DNA) with the help of a genetic material, which is self-replicating. Gene cloning duplicates only single genes of the DNA of an organism, unlike the reproductive cloning, in which the entire organism is replicated.
The recombinant DNA technology technically involves the combining of fragments of DNA from different sources, whereas the manipulation of alteration of the genetic composition of an organism is involved in genetic engineering.
The example of the recombinant DNA technology is the ligation of a piece of human DNA in to the bacterial chromosome.
The example of genetic engineering is to place the piece of recombinant DNA formed by using the recombinant DNA technology into a bacterial cell to create a bacterium.

Question 2

Describe the importance of DNA ligase in a recombinant DNA experiment. What does this enzyme do and how does its action differ from the function of restriction enzymes

Answer 2

The importance of DNA ligase enzyme in the RDT or recombinant DNA experiments is that it is used to form a phosphodiester bonds between the fragments of the DNA. The formation of phosphodiester bond is an important step in the experiments of cloning because between the sticky ends of the fragments of the DNA the hydrogen bonds are not strong enough to hold a recombinant DNA molecule together for long time or permanently.
The restriction enzymes are the enzymes that cut the DNA at recognition sequences or specific nucleotide sequences. The restriction enzymes produce two types of ends in DNA fragments, sticky ends and blunt ends. Often, the restriction enzymes are used to digest the DNA into fragments as an important step in many experiments of cloning. The restriction enzymes are used before the use of DNA ligase enzyme to join the fragments of DNA together.

Question 3

Your lab just determined the sequence of a rat gene thought to be involved in controlling the fertilizing ability of rat sperm. You believe a similar gene may control fertility in human males. Briefly describe how you could use what you know about this rat gene combined with PCR to clone the complementary human gene. Be sure to explain your experimental approach and the necessary lab materials. Also explain in detail any procedures necessary to confirm that you have a human gene that corresponds to your rat gene.

Answer 3

The cloned sequence of a gene that thought to be responsible for controlling the fertilizing capability of the rat sperm could be used to synthesize primers that probably can be used to amplify the deoxyribonucleic acid (DNA) from the human cells in an attempt to amplify the complementary genes found in humans. If the desired polymerase chain reaction (PCR) products were successfully obtained from the above experiment, the sequencing of PCR products could be done and also compared to the rat gene to search for sequences of nucleotide which are similar. This would suggest that these genes are related or not.
Also, in library screening experiments these polymerase chain reaction (PCR) products could be used as probes to find or search the full-length human gene. In northern blot analysis, these PCR products can also be used as probes to determine if the mRNA (messenger ribonucleic acid) for this gene is expressed in tissues of humans also.

Question 4

What features of plasmid cloning vectors make them useful for cloning DNA Provide examples of different types of cloning vectors and discuss their applications in biotechnology.

Question 5

If you performed a PCR experiment starting with only one copy of double-stranded DNA, approximately how many molecules would be produced after 15 cycles of amplification

Question 6

Compare and contrast genomic libraries with cDNA libraries. Which type of library would be your first choice to use if you were attempting to clone a gene in adipocyte (fat) cells that encodes a protein thought to be involved in obesity Explain your answer. What type of library would you choose if you were interested in cloning gene regulatory elements such as promoter and enhancer sequences

Question 7

Visit the Online Mendelian Inheritance in Man Site (OMIM) at the Companion Website and then click on "Search the OMIM database." Type diabetes in the search box and then click "Submit Search." What did you find Try typing 114480 in the search box. What happened this time Alternatively, search for a gene you might be interested in and see what you can find in OMIM. If the results of your OMIM search are too technical, visit the "Genes Disease" section of the NCBI site from the Companion Website then search for diabetes.

Question 8

Software analysis of DNA sequences makes it much easier for molecular biologists to study gene structure. This activity is designed to help you experience applications of DNA analysis software. Imagine that the following very short sequence of nucleotides, GGATCCGGCCGGAATT CGTA, represents one strand of an important gene that was just mailed to you for your research project. Before you can continue your research, you must find out which restriction enzymes, if any, cut this piece of DNA.Go to the Webcutter site from the Companion Website. Scroll down the page until you see a text box with the title, "Paste the DNA Sequence into the Box Below." Type the sequence of your DNA piece into this box. Scroll down the page, leaving all parameters at their default settings until you see "Please Indicate Which Enzymes to Include in the Analysis." Click on "Only the following enzymes:" and then use the drop-down menu and select BamHI. Scroll to the bottom of the page and click the "Analyze sequence" button. What did you find Is your sequence cut by Bam HI Analyze this sequence for other cutting sites to answer the following questions. Is this sequence cut by EcoRI How about SmaI What happens if you do a search and scan for cutting sites with all enzymes in the database

Question 9

Go to the molecular biology section of the Biology Project website from the University of Arizona (you can find the address on the Companion Website). Link to "Recombinant DNA Technology" and test your knowledge of recombinant DNA technology by working on the questions at this site.

Question 10

Search the Web for the company Sciona, which markets the MyCellf DNA evaluation kit for nutrigenomics. Do you think kits such as this should be used, even though they are largely based on unproven information

Question 11

What is the structural difference between a deoxyribonucleotide (dNTP) and a dideoxyribo-nucleotide (ddNTP) used for DNA sequencing

Question 12

Describe several findings of the Human Genome Project.

Question 13

Modern biology is experiencing an "omics" revolution. What does this mean