Question

28. Describe at least three uses of synthetic oligonucleotides.

29. Describe at least three ways to make a "Library"

30. Describe at least three ways to obtain DNA sequence information.

31. How do you knock out a specific gene in a mouse?

Answer 1

There are four questions asked here. As per Chegg Guidelines, I am answering the first two questions 28 and 29.

28. Synthetic oligonucleotides are sequences of nucleotides that are prepared synthetically using chemical reactions. The most widely used method to prepare these synthetic oligonucleotides is phosphoramidite chemistry.

There are many types of synthetic oligonucleotides depending on the purpose they are used for:

1. Probes: There are single stranded synthetic oligonucleotides that are designed to detect a specific sequence in the cell/solution. These are attached covalently to a fluorophore or some other molecule which can be detected. These are highly sequence specific. When bound to substrate, the fluorophore can be illuminated to radiate out fluorescence. Thus the DNA segment/RNA segment of interest can be detected.
2. Primers: These are also single-stranded oligonucleotides consisting of 15-25 basepairs that can bind to the template DNA in a PCR reaction. Based on the design, these can be made highly specific. They act as a starting point for the DNA Polymerase to initiate template-based synthesis, thereby producing many amplicons.
3. Small interfering RNA: Single-stranded RNA molecules can be injected into cells to bind to their complementary RNA molecules. This prevents the translation of this RNA and hence the protein production is halted. Thus, this can be used to manipulate the system and/or study the effect of protein in the cell. Recent advances have, however, found that the double-stranded RNA molecules are much efficient at this than their single-stranded counterparts.

29. Sequencing of larger sequences is tedious and highly error-prone. Hence the large DNA sequence is converted into a library of sequence which can then be sequenced easily with lesser errors. These library sequences can then be joined together to obtain the sequence of the larger DNA fragment.

Library generation methods vary based on the needs of the experimenter and the availability of resources. Here are listed three typical methods employed for the same:

1. Fragmentation and Adaptor Ligation: The DNA sequence is subjected to fragmentation by restriction endonucleases. The fragments are then selected for based on size. The fragments are then ligated to adaptors so that same primers can be used to amplify all the fragments. The resulted amplicons are then sequenced. The resultant sequences are screened for the overlapping fragments which can then be used to generate the sequence for the entire DNA.
2. Random primer amplification: Another method used instead of fragmentation by the RE, is the amplification of the template in a controlled manner via random primers. The random primers are usually a combinatorial library of hexamers. These primers are added one at a time to the mix and the PCR is performed with a specifed amplification time. The resultant fragments are PCR purified, adaptor ligated and then amplified before sequencing.
3. cDNA library generation: Often, the interest of the experimenter is to study the RNA sequences or the exon sequences. This is not a straightforward task, since isolation followed by direct sequencing of RNA by RNA-dependent RNA Polymerase is highly error-prone. Hence the RNA is first isolated and converted to cDNA by RNA-dependent DNA-polymerase or often known as Reverse Transcriptase. The cDNA that is obtained can then be subjected to different methods as described in the previous two methods to generate a library.