Badlis, Shannel Audrey Y.

BIO101-1_B21

cDNA Cloning

Introduction

cDNA cloning is an optimal and long-established technology in molecular biology that

produces copies of cellular mRNA (Harbers, 2008). This technique involves an mRNA population

that carries a specific mRNA for any protein expressed in the developmental stage from which the

population is isolated. This mRNA is to be isolated, and its gene will be studied. Although an

mRNA cannot be directly cloned, its DNA copy can be; it is done by reverse transcriptase and

DNA polymerase. The former prepares a single-stranded DNA copy of the mRNA, while the latter

generates the second DNA strand. The resulting double-stranded DNA is then introduced into an

appropriate vector. The usual aim of cDNA cloning is to obtain a DNA sequence that directs a

specific protein production (Borovkov et al., 1997). It can also be used to identify the mutated gene

in patients whose diseases are being studied.

In this study, protein amino acid sequences will be used to find the beta-globin gene, a

protein part of the hemoglobin that is defective in sickle-cell anemia and beta-thalassemia. A not-

very degenerate stretch of amino acids will be utilized to design a primer, which will be labeled

radioactive and used as a probe to detect clones in the cDNA library. Hybridization is the process

of allowing a labeled probe to find a DNA or RNA that it will bind to; it requires very precise

conditions to proceed. If the set conditions are incorrect, the probe may not be able to find its

complementary DNA, and it may bind to many DNAs to which it is not complementary or both.
 With this, the objectives of this study are to design an oligonucleotide corresponding to a

protein amino acid sequence, hybridize the primer to a cDNA library, and identify the hybridizing

clones among the background spots.

Methods

The initial procedure in conducting the study was designing the primers to probe the cDNA

library using a particular amino acid sequence of the beta-globin protein; the resulting primer has

a degeneracy of 128. Afterward, the labeling reaction was set by attaching a radioactive label to

the primer; 20 µL of gamma 32P-ATP was added to the tube containing the primer. Aside from

this, 7.5 µL of 10x kinase buffer and 5 µL of T4 polynucleotide kinase were also added to the

primer tube. The cDNA libraries selected for the study were the human liver and human skin.

Then, the prehybridization was set up by putting the four filters, two for each library, and

transferring 10 ml of hybridization solution into a roller bottle; the bottle was capped and placed

in the hybridization oven at 42˚C for an hour. Afterward, the bottle was removed from the oven,

and 25 µL of the probe was added to it. The bottle was again capped and put into the oven at 37˚C;

the bottle was put back on the bench after 4 hours. The next procedure done was washing the filters

by pouring off the hybridization solution in the bottle into a radioactive waste bottle, transferring

10 ml of wash solution to the bottle, and putting it back in the oven for an hour at 42˚C. Finally,

after removing the bottle from the oven, the filters were transferred to the X-ray cassette; the filter

exposed the X-ray film.

Results and Discussion

Figure 1. Filters in X-ray cassette Figure 2. cDNA X-ray image

The image of the filters placed in the X-ray cassette and the X-ray image of the cDNA is

presented in figures 1 and 2, respectively. The images on the upper and lower left of the figure are

for the 1-A and 1-B skin libraries, respectively. Meanwhile, the image on the upper right is for the

2-A liver library, and that on the lower right is for the 2-B liver one. It can be observed that the

number of clones found on the two filters for the skin and liver libraries is proportional to the

amount of RNA that codes for beta-globin in the tissue used to make the cDNA library. This

indicates that the probe is complementary to beta globin. Focusing on the filters with the skin

library, it is evident that several clones are present or aligned in the 1-A and 1-B filters. This

indicates that there is a couple of beta-globin RNA in the skin. Meanwhile, it can be observed that

there are more clones present in both the 2-A and 2-B filters, indicating that the liver is more

abundant in beta-globin RNA than the skin. These observations are aligned with how sickle-cell
anemia, caused by defective beta-globin protein, affects organs such as the lungs, liver, kidney,

and spleen; the skin may not be significantly affected by it (Lonergan et al., 2001).

Conclusion

In conclusion, the study is focused on designing an oligonucleotide corresponding to a

protein amino acid sequence, hybridizing the primer to a cDNA library, and identifying the

hybridizing clones among the background spots. The study was performed by designing primers

to probe the cDNA library, setting up the labeling reaction, incubating the labeling reaction,

selecting cDNA libraries, setting up the prehybridization, adding a probe to the roller bottle, and

washing and placing the filters in the X-ray cassette. Results showed that the probe is

complementary to beta-globin. Moreover, the X-ray image indicates that the skin and liver have

beta-globin RNA. However, the liver is more abundant in this protein than the skin.
References

Borovkov, A. Y., McClean, P. E., & Secor, G. A. (1997). Organization and transcription of the

gene encoding potato UDP-glucose pyrophosphorylase. Gene, 186(2), 293–297.

https://doi.org/10.1016/S0378-1119(96)00724-X

Harbers, M. (2008). The current status of cDNA cloning. Genomics, 91(3), 232–242.

https://doi.org/10.1016/J.YGENO.2007.11.004

Lonergan, G. J., Cline, D. B., & Abbondanzo, S. L. (2001). Sickle Cell Anemia. RadioGraphics,

21(4), 971–994. https://doi.org/10.1148/radiographics.21.4.g01jl23971