I. What are DNA Libraries?
It’s amazing to see and ponder about the exponential advancement of biotechnology and how we’ve been able to enter a new field of genomics in molecular biology! Less than 80 years ago, we just discovered DNA’s structure and now we have successfully sequenced the human genome!
Biotechnological advancements in DNA cloning and sequencing have led to the generation of DNA libraries which are exactly what they sound like as we’ll get into later! These haven’t just been created for the sake of it, but also are involved in many applications, even towards disease research!
Although a relatively low yield topic, there is another topic that we’ll discuss within the article that will have a really great overlap when talking about certain types of viruses (hint, hint!). We’ll try to keep this article short and sweet so you can get a quick understanding!
II. DNA Libraries
As their name suggests, DNA libraries refer to the collection of DNA molecules and fragments derived from a cell. There are 2 main types of DNA libraries: 1) Genomic Libraries and 2) Complementary DNA (cDNA) Libraries.
A. Genomic Libraries
These DNA libraries theoretically consist of DNA fragments which summate to the organism’s entire genome, which thus includes coding and noncoding DNA sequences.
Though these libraries contain the organism’s entire genome, there are certain characteristics of cDNA libraries that make them a little more advantageous as explained below.B. Complementary DNA (cDNA) Libraries
As indicated by the name, these libraries contain many fragments of complementary DNA (cDNA), which is DNA synthesized utilizing an mRNA molecule as a reference template! Let’s look further below to see how this occurs.
I. Generation of cDNA
As you might have already noticed, this process violates the central dogma, as we’re going from mRNA to DNA!
Here, the mRNA transcript is utilized as a template by reverse transcriptase, a RNA-dependent-DNA-polymerase, in order to generate the double stranded cDNA!
One important thing to understand is that the cDNA will ONLY CONTAIN the coding, exon regions and WILL NOT have the noncoding, introns.
Recall that mature mRNA will have its introns spliced out prior to translation as these regions do not code for any protein information!
II. Determining Gene Function
One of the benefits of having a cDNA library is the possibility of determining the function of a gene! This is relatively easy as we already have the mRNA transcript!
From here, we can translate the mRNA to a protein (or RNA) and possibly identify what function the protein has in the cell!
Similarly, you can even compare the protein structure homology with other known proteins, such as comparing the protein sequence or domain to see if the protein has homology to kinases or transcription factors!
There’s also another way cDNA libraries can help with determining gene function, which is better explained in the bridge portion of the article!
III. Bridge/Overlap
Because cDNA libraries utilize mRNA transcripts as the basis for their generation, they have an advantage over genomic libraries as they are more accurate in terms of representing gene expression in a cell. Let’s see how this applies to how we can use cDNA libraries to also help determine gene function.
I. Understanding Gene Expression
Recall that the presence of mRNA transcripts is indicative of gene expression as it confirms gene transcription! Gene expression is very dependent on cell type: though all our cells are genetically identical, all genes WILL NOT be expressed as specific cell types call for specific gene expression.
As such, cDNA libraries will be specific for a cell type, which allows us to get a better idea of the function of a gene throughits expression levels given by the cDNA library!
Here’s one way we can apply cDNA libraries to possibly determine certain gene function: we can see which genes are primarily expressed and upregulated in a normal cell and cancer cell.
We can at least say for Bcl-2, VEGF, and BCR-ABL, they all have some function in promoting cancer growth, as they’re upregulated by the cancer cell as displayed by its cDNA library.IV. Wrap Up/Key Terms
Let’s take this time to wrap up & concisely summarize what we covered above in the article!
A. Genomic Libraries
These DNA libraries consist of many DNA fragments which essentially summate the whole genome of an organism’s cell, including coding and noncoding DNA sequences.
B. Complementary DNA (cDNA) Libraries
These DNA libraries consist of many complementary DNA (cDNA) molecules, which are actually derived through utilizing a mRNA template, which opposes the central dogma!
I. Generation of cDNA
An mRNA transcript is utilized as a template to generate the complementary DNA by reverse transcriptase, a RNA-dependent-DNA-polymerase!
Note that the cDNA WILL ONLY contain the coding, exon regions and NOT the noncoding, introns. This is because prior to the mRNA being translated, the introns are spliced out so that only the coding regions remain.
II. Determining Gene Function
cDNA libraries can be helpful in possibly determining gene function! Because you already have the mRNA, it can be translated into protein and be analyzed for its protein function and be compared to see if any homology exists with other known proteins!
In addition, cDNA libraries will also be specific for cells as it’s also a measure of gene expression. You can use cDNA libraries to see what genes are (and are not) expressed in a certain cell!
V. Practice
Take a look at these practice questions to see and solidify your understanding!
Sample Practice Question 1
Given a p53 gene in a genomic library and complementary DNA (cDNA) library, what would you expect of the size of the cDNA p53 generated variant compared to the fragment in the genomic library?
A. It will be larger.
B. It will be smaller.
C. It will be the same size.
D. Cannot determine based on the information.
Ans. B
Because the p53 cDNA is generated from an mRNA transcript, it should be smaller as it only contains the exons and not the introns! Recall that the introns are spliced out before the mRNA is mature and can be translated!
Sample Practice Question 2
When reverse transcriptase acts on the mRNA transcript, it will generate the first DNA strand using the mRNA transcript as a template. Then, DNA polymerase is used to generate the second DNA strand. Which of the following best describes the first DNA strand that is generated?
A. Noncoding Strand
B. Coding Strand
C. Nontemplate Strand
D. None of the above.
Ans. A
This one is a little bit tricky but let’s try to break it down! It helps to brush up a little on DNA transcription! In transcription, the 2 DNA strands have specific terms: a coding, nontemplate strand, and a noncoding, template strand.
The noncoding, template strand will be utilized as a template to generate the mRNA transcript and thus will be complementary and antiparallel.
In this case, we’re doing the reverse: we use the mRNA transcript to generate the complementary, antiparallel strand which during transcription was the noncoding, template strand.
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