Course: SAT 170 Introductory Biotechnology

Unit 7 Reading and Multimedia Assignment

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“ DNA Library Management” by Boundless, CC BY SA 3.0

Link: https://www.boundless.com/biology/biotechnology/dna-and-biotechnology/dna-library-management/

Reading Assignment: DNA Library Management
A genomic library is a catalog of an organism's genes, created by cleaving the genome into fragments that are inserted into a vector.

Genomic libraries are a catalog of genes of a particular organism. To create a genomic library, the complete genome of an organism is cleaved into fragments and inserted into a cloning vector. Genomic library can also refer to the collection of vector molecules. With complete information in the library for a specific organism, researchers can perform a variety of experiments on the DNA. In this way they can determine the actions and interaction of separated genes along the strand. They can also compare the genomic library of healthy and unhealthy individuals of the same species to see where differences in genetic coding may have led to unexpected mutations.

In physical reality, a genomic library for humans is a collection of bacteria; the most common one is E. coli. The usable sequence of DNA from the human genome gets transferred and carried through the E. coli. The DNA is prepared by digesting it with a restriction enzyme. Then the target segments are inserted into the bacteria. This creates a basic unamplified library, where the bacteria have been allowed to multiply and create additional copies of each section of the DNA. Historically, DNA has been transferred to bacteria by using lamba phage, but these are no longer commonly used.

Artificial chromosomes are more useful in cloning larger fragments of DNA because plasmids can only contain up to 10,000 base pairs and phages are hard to work with. There are two types of artificial chromosomes: Bacterial artificial chromosome (BAC) and yeast artificial chromosomes (YAC). Artificial chromosomes can contain anywhere from 300,000 (BAC) to 1,000,000 (YAC) base pairs, effectively reducing the amount of runs needed for a large fragment to be analyzed. Because these chromosomes are more useful in cloning larger fragments of DNA, it is easier and quicker to clone and transform genes. Artificial chromosome vectors also make it easier to store through bacterial cells rather than mammalian cells.

While a gene library is a catalog of all the genes in a given organism, it does not give any information as to which of these genes are expressed at a given time. In many cases, scientists are interested in identifying only genes that are "turned on" in the organism. To do this, they need to look more specifically at the genes that are currently being transcribed. When a gene is activated, cellular machinery begins to copy certain segments of that gene. The resulting product is known as messenger RNA (mRNA), which is the body's template for creating proteins.

To determine which genes are turned on and which are turned off in a given cell, a researcher must first collect the messenger RNA molecules present in that cell. The researcher then labels each mRNA molecule by using a reverse transcriptase enzyme (RT) that generates a complementary cDNA to the mRNA. After the reverse transcript is made, the mRNA is degraded and DNA polymerase will synthesize a complementary strand from the reverse transcript. This strand is called complementary DNA, or cDNA.

If a particular gene is very active, it produces many molecules of messenger RNA. Therefore, it produces more labeled cDNAs that hybridize to the DNA on the microarray slide and generate a very bright fluorescent area. Genes that are somewhat less active produce fewer mRNAs and less labeled cDNAs, which results in dimmer fluorescent spots. If there is no fluorescence, none of the messenger molecules have hybridized to the DNA, indicating that the gene is inactive. Researchers frequently use this technique to examine the activity of various genes at different times.

A cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. Similarly, tissue specific cDNA libraries can be produced. In eukaryotic cells the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell.

cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. However, a cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries provide much more detailed information about the organism, but are much more resource-intensive to generate and maintain.