cDNA is complementary DNA synthesized from an RNA transcript. It is a more stable form than RNA, making it suitable for long-term storage and manipulation.
RNA is isolated and reverse transcribed into cDNA for various applications, particularly in gene expression studies, as RNA itself is highly susceptible to degradation.
Basics of cDNA Production
Definition of cDNA: cDNA is synthesized by reverse transcribing mature mRNA. It represents the coding sequences of genes without intronic sequences, reflecting the spliced transcript present in eukaryotic cells.
Purpose of cDNA production: To generate a stable DNA form of a specific mRNA for downstream applications like cloning, sequencing, or gene expression analysis. This allows researchers to study genes as they are expressed in a particular cell type or condition.
Isolation of RNA
Total RNA vs. mRNA: Total RNA includes all RNA types (mRNA, tRNA, rRNA) from the tissue. rRNA constitutes the majority of total RNA (up to 80-90%), while mRNA makes up only 1-5%.
Isolating specific mRNA: Specific mRNA is selectively isolated from total RNA, typically through oligo-dT affinity chromatography which exploits the poly-A tail of eukaryotic mRNA. Reverse transcription is then performed to obtain cDNA.
Process of isolation: Total RNA is initially extracted from tissues using methods like guanidinium thiocyanate-phenol-chloroform extraction or spin column-based kits. Subsequently, specific mRNA targeting involves using primers, commonly oligo-dT primers, that bind specifically to the poly-A tail present at the 3' end of intact eukaryotic mRNAs.
Reverse Transcription Process
Use of primers: Oligo-dT primers, typically between 15-20 bases long, are commonly used for eukaryotic mRNA as they anneal to the poly-A tail. Alternatively, random hexamers or gene-specific primers can be used.
Limitations in bacterial RNA: Bacteria do not have poly-A tails; hence, different methods for cDNA synthesis are needed, often involving random hexamers or gene-specific primers.
Reverse transcriptase: An enzyme derived from RNA viruses (retroviruses) that synthesizes DNA from an RNA template. Common examples include M-MLV (Moloney Murine Leukemia Virus) and AMV (Avian Myeloblastosis Virus) reverse transcriptases.
Reverse transcriptase function: It synthesizes cDNA, extending in the 5′ to 3′ direction, using an RNA template and a primer. The resulting product is an RNA-DNA hybrid molecule.
Creating Double-Stranded cDNA
Single strand to double strand: After forming a single-stranded cDNA from the RNA template, the RNA template is removed using RNase H, an enzyme that selectively degrades the RNA strand in an RNA-DNA hybrid, leaving behind the single-stranded cDNA.
Filling in the missing strand: A standard DNA polymerase (such as E. coli DNA polymerase I, which can utilize the exposed single-stranded cDNA as a template and the remaining RNA fragments as primers) then extends the incomplete strand to form double-stranded cDNA. This process often involves the formation of a hairpin loop at the 3' end of the first strand, which serves as a self-primer for the second strand synthesis.
Incomplete sections: A portion of the RNA may never be fully replicated, similar to problems in linear DNA replication where end-sequences (telomeres) might be missing, leading to incomplete cDNA products, especially at the 5' end of the original mRNA.
Applications of cDNA
Gene expression analysis: cDNA synthesis allows for analyzing gene expression levels in different tissues, developmental stages, or disease states by comparing cDNA collections. Techniques like quantitative PCR (qPCR) or RNA sequencing (RNA-Seq) rely on cDNA for this purpose.
Sequencing: After synthesizing cDNA, researchers can sequence these cDNAs to determine which genes are expressed in specific tissues and to identify splice variants or mutations within the coding regions.
Amplification of cDNA via PCR
Need for amplification: The initial cDNA yield from reverse transcription is often low, necessitating amplification to obtain sufficient material for subsequent analysis or cloning experiments.
PCR definition: Polymerase Chain Reaction (PCR) allows the exponential amplification of specific DNA sequences. It is a highly sensitive and specific method.
History of PCR: Developed by Kary Mullis in the 1980s, drastically reducing the time and effort required for gene analysis from years to weeks or even hours.
Impact of PCR: Before PCR, cloning a gene for study could take several years; post-PCR, this could be achieved in a matter of weeks, revolutionizing molecular biology.
Essentials for PCR
Primers: Specific oligonucleotide probes (typically 18-30 bases long) are required to initiate amplification, based on the known sequence of the target gene's flanking regions. Primers cannot contain ambiguous sequences and should be designed to avoid primer-dimers and significant secondary structures. They typically have a GC content of 40-60%.
Temperature cycling: The process requires alternating heating (to denature DNA) and cooling (to allow for primer annealing). A complete PCR cycle consists of three main steps:
Denaturation at approximately 95extoextC (to separate DNA strands).
Annealing at 50ext−60extoextC (to allow primers to bind to complementary sequences).
Extension at 72extoextC using thermostable DNA polymerases like Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus.
Summary of PCR Steps
Denaturation: DNA is heated to 94ext−98extoextC for 15ext−30 seconds to separate the double-stranded DNA template into single strands by breaking hydrogen bonds.
Annealing: The temperature is lowered to 50ext−65extoextC for 15ext−60 seconds to allow the forward and reverse primers to bind (anneal) to their complementary sequences on the opposing single strands.
Extension: At 72extoextC (the optimal temperature for Taq polymerase), Taq polymerase extends the new DNA strand from the 3′ end of each primer, synthesizing a complementary strand to the template DNA at a rate of approximately 1000 base pairs per minute.
This cycle is repeated typically 35ext−45 times to exponentially amplify the target DNA, resulting in millions to billions of copies.
Product Isolation and Cloning
Gel electrophoresis: Used to purify and visualize amplified products from the PCR process. DNA fragments are separated based on size through an agarose gel matrix and then stained (e.g., with ethidium bromide) for visualization under UV light.
Inclusion in expression plasmids: The goal is to insert amplified cDNA into plasmids suitable for expression in E. coli or other host organisms. This often involves cutting both the plasmid and the PCR product with restriction enzymes that generate compatible ends.
Requirements for Plasmid Design
Origin of replication (Ori): Essential for plasmids so that they replicate independently alongside the bacterial chromosome, ensuring multiple copies of the plasmid are maintained in each host cell.
Selectable markers: Include antibiotic resistance genes (e.g., ampicillin resistance, kanamycin resistance) to allow for the selection of positive transformants. Only bacteria that have successfully taken up the plasmid will survive and grow on selective media containing the antibiotic.
MCS (Multiple Cloning Site): Also known as a polylinker, this is a short segment of DNA containing several unique restriction enzyme recognition sites, allowing for the flexible insertion of target genes into the plasmid.
Restriction Enzymes and Ligation
Restriction endonucleases: Enzymes that cut DNA at specific palindromic sequences (recognition sites), often resulting in sticky (overhanging) or blunt ends. Different enzymes recognize different sequences.
Sticky ends: Overhanging single-stranded sequences (e.g., 5′ or 3′ overhangs) that allow for easier and more efficient ligation of compatible DNA fragments due to transient hydrogen bonding between complementary bases. Example: EcoRI generates a 5′ overhang (G^AATTC).
Blunt ends: Straight cuts that leave no overhanging bases. Blunt-ended fragments can be joined to any other blunt-ended fragment, but this process is less efficient than sticky-end ligation, as it does not involve complementary base pairing.
DNA ligase: Facilitates the joining of DNA fragments by catalyzing the formation of phosphodiester bonds between the 5′ phosphate and 3′ hydroxyl groups of adjacent nucleotides, repairing the nicks in the sugar-phosphate backbone.
Transforming E. coli with Plasmids
Methods of introduction:E. coli can take up plasmids by chemical transformation (treatment with ice-cold calcium chloride solution followed by a brief heat shock at 42extoextC to increase membrane permeability) or electroporation (using a brief high-voltage electric pulse to create transient pores in the cell membrane).
Selection of successful transformants: Bacteria that have taken up plasmids are grown on culture media containing antibiotics corresponding to the selectable marker gene on the plasmid. This eliminates non-transformed cells, ensuring only cells containing the desired plasmid grow.
Summary of the Transformation Process
Culture and incubation: After transformation, cells are plated on selective agar media and incubated (typically overnight at 37extoextC). During this period, only those cells carrying plasmids with the antibiotic resistance gene will survive and divide.
Outcome: Successful transformation results in the formation of colonies, each derived from an individual E. coli cell that has taken up and retained the recombinant plasmid. These colonies can then be picked for further analysis and propagation.