I had a wonderful opportunity to learn about DNA sequencing and bioinformatics while attending the virtual nine week Waksman Institute Summer Experience program. We learned how to isolate the complementary DNA or cDNA fragments from the duckweed (Landoltia punctata) plant, using techniques like reverse transcriptase, plasmid vectors, polymerase chain reaction (PCR), gel electrophoresis etc. We used bioinformatics tools like BLAST, etc. to compare the sequenced DNA with known sequences stored in online repositories like GenBank. We also learned how to determine if the cDNA codes for a protein using the ORF Toolbox and look for similar proteins in other organisms. The duckweed plant grows on water in lakes and wetlands. It’s being researched as a potential food source and in bioremediation. With the help of the vWISE team, I submitted clones W418.20 (Landoltia punctata clone W418.20, 2021) and W417.20 (Landoltia punctata clone W417.20, 2021) to GenBank. The clone W418.20 codes for a protein that is similar to acid phosphatase/vanadium-dependent haloperoxidase-related protein.
Part one of the four part series starts off with the creation of the complementary DNA or cDNA library. A cDNA library contains random DNA fragments isolated from an organism which can be stored for future research. The cDNA are genes that are expressed at specific times by the cells of the organisms. The cDNA is reverse transcribed from the mRNA extracted from the organism (Hoy, 2018). The cDNA codes for proteins which are useful in research. The genomic library, like the ones generated from the Human Genome Project, contains large DNA fragments which can contain both coding and non-coding regions whereas cDNA libraries only contain DNA fragments that code for proteins.
Create cDNA library of the organism
There are various protocols or procedures to isolate RNA from the organisms, which depends on type of organism and the part of the organism used. The organism first needs to be homogenized. Homogenization is the breaking up of the organism matter into smaller particles and to lyse cells (Shechter, 2019). Lysis is the breaking up of the membranes of the cells to release contents of the cells. One way of doing this is by freezing the samples of the organism in nitrogen and then grounding the samples using mortar. The contents will contain proteins, DNA, RNA, debris, cell walls etc (Johnson M., et al., 2012). To remove the debris, the contents are then centrifuged where the debris is pelletized. DNA and RNA fragments are separated from the proteins in the centrifuged solution using phenol. Then the RNA is precipitated out of the remaining solution using lithium chloride and ethanol. Sometimes DNAse is used to remove DNA leaving behind RNA.
Purify mRNA from RNA
The RNA precipitated above contains different types of RNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and others. For the cDNA library, we are interested in mRNA since these code for proteins. By passing the “total” RNA over an oligo deoxythymine (oligo-dT) column, mRNA will stick to the column while other components pass through. This is because mRNA has a tail of adenosine nucleotides called poly-A tail and this hybridizes with the oligo-dT on the column causing the mRNA to stick to it (Purification of Messenger RNA by Affinity Chromatography on CIMmultusTM Oligo DT Column – BIA Separations, 2021). Hybridization is a process of combining two complementary single strands of DNA or RNA molecules to form a double strand by pairing bases. The column is then washed to remove any contaminants. The mRNA is then eluted or removed from the column by washing it with a buffer.
The image below (taken from Nature Education and adapted from Pierce, Benjamin. Genetic: A Conceptual Approach, 2nd ed.) shows the the steps for mRNA isolation as part of the generation of cDNA from mRNA.
The video below by Kenneth Francis Rodrigues shows an example of the process.
Reverse transcribe the mRNA to get cDNA
The messenger RNA or mRNA is a single strand RNA molecule that is the complementary of a strand of DNA in a gene. It carries this information to the cell which creates proteins. In order to generate the complementary DNA or cDNA from the mRNA, special enzymes called Reverse Transcriptase or RT are used (Pray, 2008). The reverse transcciption goes through certain steps. First, oligo (dT) primers are added to the mRNA. This hybridizes or anneals the single strand mRNA with the primer at the ends. The ends also has a special site which is later used to insert cDNA strand onto a plasmid for amplification. The RT enzymes then synthesizes the double strand cDNA using the mRNA. This contains the mRNA and the cDNA. This stage is called “first strand synthesis“. Next, the RNA part is removed from the above strand by hydrolyzing (breaking down) it with sodium hydrozide.
The second strand of DNA is then synthesized using a DNA polymerase enyzme and oligonucleotide primers. The primers also contain a special site for inserting the DNA into a plasmid. This is the “second strand synthesis“. Thus, the cDNA is generated from the mRNA.
The image below (taken from Nature Education and adapted from Pierce, Benjamin. Genetic: A Conceptual Approach, 2nd ed.) shows the the steps for the reverse transcription as part of the generation of cDNA from mRNA.
Amplify cDNA to create cDNA library
The amount of cDNA generated is too small to perform any analysis. The amount of the cDNA needs to be amplified. One way of doing this is with the use of plasmids and some help from bacteria like E. Coli. Plasmids are small circular strands of DNA that bacteria can clone and multiply. This molecular cloning will also multiply any of the ligated alien DNA fragment thereby amplifying the amount of the DNA under study. Newer and faster methods use Polymerase Chain Reaction (PCR) method (Pray, 2008) which will be described in part 2 of this post.
The cDNA fragments are cut up or digested at the location of the special sites using special enzymes called restriction digest. The average lengths of the cut up fragments will be around 64 kilo base pairs (64 kbp). The cut up DNA fragments are then ligated or pasted onto plasmids using an enzyme called DNA ligase. The special plasmid used here is pTriplEX2.
The plasmids containing the ligated cDNA fragments are then transformed into E. Coli bacteria. The bacterial transformation is the process of incorporating foreign DNA into the cells of a bacteria such that the bacteria expresses the acquired DNA (Bacterial Transformation and Competent Cells–A Brief Introduction | Thermo Fisher Scientific – NL, n.d.). The E. Coli bacteria is treated with calcium and exposed to brief high temperature so that it can accept the plasmids.
Not all plasmids will contain the target cDNA and not all bacterial cells will contain any of the plasmids. The plasmids contain an engineered site that makes it resistant to ampicillin. In order to only grow the bacteria with the plasmids, the bacterial culture is plated on Luria-Bertani (LB)-agar media, which is a food source to grow the bacterial colonies. These plates are then treated with ampicillin to select the transformed cells. The bacterial cells that contain ampicillin resistant plasmids will grow while the others die out. However, not all these transformed bacterial cells contain the target cDNA. To identify the ones that have the target cDNA, the plasmids have a color screen gene called LacZ. This gene creates an enzyme called β-galactosidase. Plasmids containing the cDNA insert interferes with the gene’s ability to produce this enzyme. When the bacterial cultures are treated with a solution called X-gal, the culture with the LacZ gene will appear blue whereas the culture with the plasmids containing the inserts will appear white (Blogger, 2019). The white colonies are collected and are stored as cDNA libraries for future research.
The diagram below (taken from ThermoFisher Scientific) shows the transformation of the bacteria and their phenotype (behavior) depending where they are carrying the insert or not.
The image below (taken from AG Scientific) shows an E. Coli bacterial culture with blue and white colonies, the white colonies containing the plasmids with the recombinant cDNA insert.
While the cCNA library was already created for this course, I’m very interested in actually performing these steps in a lab in the future. Part 2 specifies the steps to amplify the cDNA from a sample taken from cDNA library and prepare it to be sequenced.
Landoltia punctata clone W418.20 acid phosphatase/vanadium-dependent haloperoxidase-related protein-like, mRNA sequence. Jaison, C., Vershon, A. and Mead, J., NCBI, May 18, 2021. Accession# JZ984547 https://www.ncbi.nlm.nih.gov/nuccore/JZ984547.1
Landoltia punctata clone W417.20, mRNA sequence. Jaison, C., Vershon, A. and Mead, J., NCBI, May 18, 2021. Accession# JZ984546 https://www.ncbi.nlm.nih.gov/nuccore/JZ984546.
Hoy, M. A. (2018). Insect Molecular Genetics. Elsevier Gezondheidszorg.
Shechter, D. (2019, November 21). What is the Purpose of Homogenization? BEE International. https://www.beei.com/blog/purpose-of-homogenization
Johnson M., Carpenter E., Tian Z., Bruskiewich R., Burris J., Carrigan C., et al. (2012) Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes. PLoS ONE 7(11): e50226. https://doi.org/10.1371/journal.pone.0050226
KENNETH FRANCIS RODRIGUES -. (2020, June 26). Plant RNA Extraction with Qiagen RNEasy Kit and Subtitles [Video]. YouTube. https://www.youtube.com/watch?v=BIQU8HU1xms
Purification of messenger RNA by affinity chromatography on CIMmultusTM Oligo dT column – BIA Separations. (2021). Sartorius BIA Separations. https://www.biaseparations.com/en/library/application-notes/1037/purification-of-messenger-rna-by-affinity-chromatography-on-cimmultustm-oligo-dt-column
Pray, L. (2008) The Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes. Nature Education 1(1):94 https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html
Bacterial Transformation and Competent Cells–A Brief Introduction | Thermo Fisher Scientific – NL. (n.d.). Https://Www.Thermofisher.Com/. Retrieved June 7, 2021, from https://www.thermofisher.com/nl/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/competent-cell-basics.html
Blogger, A. G. (2019, October 7). X-GAL: Cloning, Protein-protein Interactions, and Water Testing for E. coli. AG Scientific Blog. https://agscientific.com/blog/2012/11/x-gal-cloning-protein-protein-interactions-and-water-testing-for-e-coli/