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Complementarity of cDNA


Strictly speaking, what is the definition of cDNA? This confuses me, since usually it is said to refer to DNA that is complementary to mRNA. Is this correct? Is it restricted to mature mRNA?

I also find directionality very confusing. As I understand it, mRNA has a sequence equivalent (less introns, after splicing) to the so-called 'coding strand' of DNA. Does this mean that cDNA made from mRNA, at least before treated with a DNA polymerase, is also complementary to the original genomic DNA? Is this the better definition (I have seen this used as well, though the first definition seems far more common).

Lastly, as I understand it each chromosome has a forward and reverse strand, defined by convention, but that the 'coding' strand of a given gene is random. This seems counter-intuitive… is it just because both strands are equivalent? Does it introduce complications in terms of the cell 'knowing' which strand is the coding strand for a gene?

The most upvoted reply to this question (http://www.biostars.org/p/3423/) confused me even further. I feel the same way as 'Onefishtwofish' does, in the replies. Can anyone clarify?


It's a good question and has confused me as well.

A standard definition of cDNA is that it is a double stranded (ds) product of mature mRNA. I suppose it is possible to get a nascent RNA strand copied into cDNA, but I've never heard of that being done.

Because cDNA is dsDNA, the original strand is made from your mRNA as the complementary strand, but then you dispose of the RNA and make the second strand to get a stable, reproducable molecule. The total dsDNA molecule is what we refer to as cDNA (not a single strand of this material).

consider this simplified DNA molecule:

ATG-INTRON-CTCTAG

TAC-INTRON-GAGATC

this could be transcribed into the following mRNA (corresponding to Met-INTRON-Leu-Stop):

AUG-INTRON-CUCUAG

before nuclear export the intron is removed, making mature mRNA:

AUGCUCUAG

you make cDNA from this:

ATGCTCTAG

TACGAGATC

So, you have both a coding and a complementary strand in your cDNA.

As for directionality on the chromosome, genes can be in either direction (meaning that they can use either strand as the coding strand), but remember that the forces that contribute to transcription are just locally determined (assembly of transcription factors, etc) and pay no heed to what direction they're going or strand they are on relative to any other gene. The strands are labeled as positive and negative, but that's just a reference for orientation. A good place to browse the human (or other species) genome is the NIH's map viewer. Click on a chromosome to see the genes mapped to it. Note the column labeled 'O' is for orientation.


3.6: cDNA

  • Contributed by Ross Hardison
  • T. Ming Chu Professor (Biochemistry and Molecular Biology) at The Pennsylvania State University

CDNA clones are copies of mRNAs

Construction of cDNA clones involves the synthesis of complementary DNA from mRNA and then inserting a duplex copy of that into a cloning vector, followed by transformation of bacteria (Figure (PageIndex<1>)).

Figure (PageIndex<1>): Making cDNA clones

a. First strand synthesis: First, one anneals an oligo dT primer onto the 3' polyA tail of a population of mRNAs. Then reverse transcriptase will begin DNA synthesis at the primer, using dNTPs supplied in the reaction, and copy the mRNA into complementary DNA, abbreviated cDNA. The mRNA is degraded by the RNase H activity associated with reverse transcriptase and by subsequent treatment with alkali.

b. Second strand synthesis: For the primer to make the second strand of DNA (equivalent in sequence to the original mRNA), one can utilize a transient hairpin at the end of the cDNA. (The basis for its formation is not certain.) In other schemes, one generates a primer binding site and uses a primer directed to that site one way to do this is by homopolymer tailing of the cDNA followed by use of a complementary primer. Random primers can also be used for second strand synthesis although this precludes the generation of a full-length cDNA (i.e. a copy of the entire mRNA). However, it is rare to generate duplex copies of the entire mRNA by any means.

DNA polymerase (e.g. Klenow polymerase) is used to synthesize the second strand, complementary to the cDNA. The product is duplex cDNA.

If the hairpin was used to prime second strand synthesis, it must be opened by a single‑strand specific nuclease such as S1.

c. Insertion of the duplex cDNA into a cloning vector:

One method is to use terminal deoxynucleotidyl transferase to add a homopolymer such as poly-dC to the ends of the duplex cDNA and a complementary homopolymer such as poly-dG to the vector.

An alternative approach is to use linkers these can be employed such that a linker carrying a cleavage site for one restriction endonuclease is on the 5' end of the duplex cDNA and a linker carrying a cleavage site for a different restriction endonuclease is on the 3' end. (In this context, 5&rsquo and 3&rsquo refer to the nontemplate, or "top" strand.) This allows "forced" cloning into the vector, and one has initial information about orientation, based on proximity to one cleavage site or the other.

The cDNA and vector are joined at the ends, using DNA ligase, to form recombinant cDNA plasmids (or phage).

d. The ligated cDNA plasmids are then transformed into E. coli. The resulting set of transformants is a library of cDNA clones.


Recombinant DNA and Biotechnology

Complementary DNA

Complementary DNA (cDNA) is synthesized in the laboratory from messenger RNA ( Fig. 18-3 ). cDNA is not genomic DNA, because the transcript of genomic RNA has been processed (i.e., it lacks promoters and introns). The enzyme reverse transcriptase (see Chapter 15 ) is used to synthesize double-stranded DNA that is a complementary copy of the mRNA. The addition of linker sequences to the end of this DNA, which contain the restriction site, followed by treatment with a restriction enzyme, produces a cDNA preparation with cohesive ends ready for insertion into a vector. A preparation of cDNA represents the genes that were actively being expressed in a cell, an organ, or a whole organism at the time of harvesting and is called a cDNA library.


Genomic DNA libraries

Size of some genomes and chromosomes:

  • The human genome contains approximately 50,000 unique genes within 3-4 billion base pairs of DNA , scattered about in 23 pairs of chromosomes .

Fragmentation of genomic DNA for library construction

Restriction endonuclease digestion

  • A six-cutter (e.g. Eco RI) will cut on average every 4.1 Kb. Complete digestion of human DNA with this type of enzyme will result in approximately 1 x 10 6 unique fragments.
  • What is the probability of finding a clone within a given library?

The exact probability of having any given DNA sequence in the library can be calculated from the equation

N = ln(1 -P)/ln(1 - f) P is the desired probability f is the fractional proportion of the genome in a single recombinant N is the necessary number of recombinants

For example, how large a library (i.e. how many clones) would you need in order to have a 99% probability of finding a desired sequence represented in a library created by digestion with a 6-cutter?

N = ln(1 - 0.99)/ln(1 - (4096/3x10 9 )) N = 3.37 x 10 6 clones

Thus, from this type of analysis we can see that we need a technology which will allow us to achieve the following:

  1. Stable insertion of relatively large DNA fragments into our cloning vector
  2. High efficiency of insertion and the ability to handle large numbers of clones
  • For example, when plating E. coli colonies on a 3" petri plate, the maximum practical density to allow isolation of individual colonies is about 100-200 colonies per plate.
  • If we were to try to plate our library of 3.37 x 10 6 in such a way would need about 22,500 plates .
  • Not only that, but such large DNA fragments are not well tolerated in typical E. coli cloning vectors such as pBR322.

Bacteriophage lambda vectors are commonly used for construction of genomic libraries

Bacteriophage l is an E. coli phage with a type of icosahedral phage particle which contains the viral genome:

Figure 3.6.6: Bacteriophase l

  • During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.
  • It was found that internal regions of the phage genome, which were not essential to phage replication, could be removed and replaced with DNA of interest.
  • This hybrid DNA could be efficiently packaged, and form an infective phage.

Figure 3.6.7:Creation of ineffective phage

The advantages of this type of system vs plasmids like pBR322 are:

  1. The phage genome is able to package efficiently with DNA inserts as large as 20 Kb.
  2. Furthermore, the packaged phage are highly infectious and infect E. coli at a much higher efficiency than plasmid transformation methods.

Incomplete Digestion of Genomic DNA will allow identification of sequence overlaps

Complete digestion with an endonuclease will result in a library containing no overlapping fragments:

However, incomplete digestion will result in a library containing overlapping fragments:

  • Thus, the sequence information obtained from one clone will allow the isolation of clones containing neighboring (overlapping) sequence information.
  • This can allow large contiguous stretches of sequence information to be obtained (" Chromosome Walking ").

Probing libraries

Once a library (cDNA or genomic) has been constructed we want to be able to identify clones which contain DNA of interest.

  • For example, from protein sequence information we can deduce possible stretches of the corresponding DNA sequence (there will however be ambiguity due to the degeneracy of codons).
  • If we can synthesize an oligonucleotide complementary to our DNA sequence of interest we can use it to specifically hybridize to the appropriate clone in our libraray (i.e. to probe our library).

In standard methodologies the oligonucleotide is phosphorylated at the 5' end with radiolabeled g 32 P-ATP and T4 polynucleotide kinase.

  • The probe is then incubated with individual phage plaques which have been fixed onto nitrocellulose and their DNA denatured by treatment with base.
  • If the plaque contains complementary DNA to to probe sequence, the probe will hybridize.
  • If the nitrocellulose (containing many individual plaques) is exposed to x-ray film, only those plaques with hybridized probe will show up (as a dark spot):

Figure 3.6.8:Radiolabeled plaque

Note that its important to keep track of the orientation of the nitrocellulose in relationship to the x-ray film (usually radioactive ink is used to identify the nitrocellulose orientation).

False positives

If we are designing DNA probes from protein sequence information we will have possible ambiguity in our deduced DNA sequence used for the design of the probe.

  • Usually 14-24mer oligonucleotides are used as probes, a 14-24mer probe means we need a stretch of 5-8 amino acids in the polypeptide.
  • Given the choice, the best amino acid sequences to look for in a polypeptide are those with low codon degeneracy (see above).
  • Thus, we would look for a short stretch of polypeptide sequence hopefully containing Met or Trp , and with the remaining amino acids comprising either Phe, Tyr, His, Gln, Asn , Lys, Asp, Glu or Cys .
  • Regions including Leu, Arg or Ser are to be avoided (6 codons each).

During oligonucleotide synthesis multiple bases will be incorporated at ambiguous positions.

  • Thus our probe will actually be a mixture of oligonucleotides.
  • The higher the degeneracy, the greater the posibility of "false positives", i.e. clones which hybridize but are unrelated to the actual sequence we want.
  • Positive clones are sequenced and the deduced amino acid sequence is compared to our polypeptide sequence information to identify correct clones.

Antibodies (Immunoglobulins)

If the particular vector, or phage, used to construct a cDNA library contains a promoter region upstream of the insertion site we may be able to screen for desired clones by looking for expression of the protein of interest .

  • In this case, we need an assay which is both sensitive (we will not be producing a lot of protein) and specific (we want to minimize any false positives).
  • One of the best assays, which is both sensitive and specific, makes use of antibodies.

Antigen, antibody, epitope

One of the defense mechanisms of vertebrates is the ability to distinguish between self and non-self molecules.

  • Thus, if a foreign molecule (either from another species or sometimes from another individual within a species) invades a vertebrate organism, the immune system functions to learn to identify that molecule.
  • In future invasions by the same molecule, the organism mounts a defense against it by producing specific antibodies which recognize and bind to the foreign antigen.
  • When antibodies bind to antigen certain white blood cells (macrophages and monocytes) recognize the invading body as foreign and respond by destroying it.

Antibodies are 'Y' shaped molecules which contain two identical heavy chains, and two identical light chains.

  • The stem of the 'Y' comprises the Fc (constant) domain , and the 'arms' of the 'Y' comprise the Fab (variable) domains .
  • Antigens bind to the complementarity-determining regions (CDR's) located at the ends of the Fab domains.

Figure 3.6.9:Antibody structure

Antibodies are synthesized by B lymphocytes. Each B lymphocyte is capable of producing a single type of antibody directed against a specific structural determinant, or epitope, on an antigen.

  • Thus, an immune response to a protein antigen may result in a population of B lymphocytes each producing antibodies which recognize a different structural determinant of the foreign protein.
  • An epitope may be a contiguous region of 5 or 6 amino acids in the foreign polypeptide, or the epitope may comprise a half dozen or so amino acids brought in juxtaposition in the native protein, yet widely spaced in the polypeptide sequence.
  • Thus, some antibodies will recognize native and denatured forms of a foreign protein equally well, while other antibodies may only recognize one or the other.

If the protein of interest has been purified it can be used to induce an immune response in a host animal .

  • Typical host animals include mouse, chicken, rabbit, goat, sheep, horse and occasionally, human.
  • After an initial immunization, followed by one or more booster shots, the B lymphocytes of the host animal may produce antibodies directed against the antigen.
  • The antibodies can be be purified from blood samples withdrawn from the animal. Such preparations of antibodies are said to be polyclonal.
  • This refers to the fact that the antibodies present are from a collection of different B lymphocytes and thus will recognize a variety of different epitopes on the antigen protein.
  • The ability to isolate antibodies from blood samples means that the host animal does not need to be destroyed.
  • Of course, the size of the animal determines how much antibodies one can obtain. For example, a rabbit can provide 5 mls of blood every two weeks, a mouse provides significantly less, while a horse can provide quite a bit more.

An antibodiy isolated from a single B lymphocyte cell population is termed monoclonal.

  • It recognizes a single epitope on the antigenic protein.
  • Antibody producing B lymphocytes can be isolated from the spleen or from lymph nodes. However, they have a finite lifespanin culture, i.e. they will undergo a certain number of cell divisions and then die.
  • These cells can, however, be fused with immortal (cancerous myeloma) lymphocytes to produce a hybridoma cell.
  • Such a cell is immortal like the myeloma, and produces a specific antibody from the B lymphocyte. The ability to grow indefinitely in culture allows the isolation of useful amounts of specific monoclonal antibodies.

Sometimes immunizing with the protein of interest is problematic: appropriate amounts of purified material cannot be produced, or the protein is itself toxic at the dosage level necessary to produce an immune response.

  • If partial sequence information is known, then large amounts of polypeptides representing short fragments of the protein, can be synthesized and used to immunize the animal.
  • Often these polypeptides are covalently attached to a carrier protein (typically serum albumin) to enhance the antigenic response.
  • Antibodies produced against such peptides will recognize only epitopes within the polypeptide. Thus, even polyclonal antibodies would be quite limited in their epitope recognition.

As with radiolabeled oligonucleotides, antibodies can be used to identify library clones which contain a cDNA of interest. This method would of course rely upon a host vector or phage which contains a promoter upstream from the site of insertion of the genomic DNA.


Complementary DNA (cDNA)

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Next, we must convert the RNA into DNA. We use an enzyme called “reverse transcriptase” to create a complementary DNA ( cDNA ) sequence from the RNA fragment. This creates hybrid molecules that are a combination of RNA and cDNA.

cDNA. mRNA is isolated from an organism of interest. The single – stranded portion of the loop is cut with an S1 nuclease, and the result is a double- stranded cDNA copy of the mRNA. Note that this cDNA will include only the exon portions of the gene, and not the introns, which were spliced out of the mRNA template.


Shop the Largest Tissue cDNA Selection in the Market

BioChain’s cDNA samples are synthesized using total RNA isolation at the facility with modified techniques to ensure consistency. cDNA undergoes both visual inspection detecting intact bands of ribosomal DNA, and tested by purity with a spectrophotometer. The first strand is synthesized using MMLV reverse transcriptase with low RNase H activity, with an oligo dT primer to ensure presence of the entire cDNA.

Sources originate from a variety of animal, plant matter, and human/fetal tissue (including healthy and diseased organs). Documentation on clinical history of tissues is available. The cDNA can be used for PCR, gene discovery, analysis or mRNA, and cloning among others.


Step 1. Prepare sample

RNA serves as the template in cDNA synthesis. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling.

Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. Best practices to prevent degradation of RNA include wearing gloves, pipetting with aerosol-barrier tips, using nuclease-free labware and reagents, and decontamination of work areas.

To isolate and purify RNA, a variety of strategies are available depending on the type of source materials (e.g., blood, tissues, cells, plants) and goals of the experiments. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Product highlights

Troubleshooting tips

  1. Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  2. Store RNA in an EDTA-buffered solution to minimize nonspecific cleavage by nucleases that have metal ion cofactors.
  3. Use water that is certified nuclease-free or treated with DEPC (diethylpyrocarbonate) to ensure the absence of RNase.
  4. Assess the integrity of RNA by gel electrophoresis or microfluidics.

Prevalance

Reverse transcriptases have been identified in many organisms, including viruses, bacteria, animals, and plants. In these organisms, the general role of reverse transcriptase is to convert RNA sequences to cDNA sequences that are capable of inserting into different areas of the genome. In this manner, reverse transcription contributes to (Figure 2):

  • Propagation of retroviruses—e.g., human immunodeficiency virus (HIV), Moloney murine leukemia virus (M-MuLV), and avian myeloblastosis virus (AMV) [1,2]
  • Genetic diversity in eukaryotes via mobile transposable elements called retrotransposons [4]
  • Replication of chromosomal ends called telomeres [5,6]
  • Synthesis of extrachromosomal DNA/RNA chimeric elements called multicopy single-stranded DNA (msDNA) in bacteria [7,8]

Figure 2. Roles of reverse transcriptase in biological systems. (A) Viral RNA is reverse-transcribed for integration into the host genome. (B) In retrotransposition, an RNA intermediate is reverse-transcribed to insert DNA copies into other areas of the genome. (C) Telomerase reverse transcriptase (TERT) uses RNA as a template to elongate and maintain eukaryotic chromosome ends. (D) Reverse transcription is an intermediate step in the formation of multicopy single-stranded DNA (msDNA) in bacteria.


Complementarity of cDNA - Biology

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Almost every cell in the body has the same DNA, but different cell types, such as neurons and muscle cells, express different genes because only certain genes are transcribed into messenger RNA, or mRNA, in each cell. In the laboratory, mRNAs can be used as a template to synthesize complementary DNA, cDNA, to study gene expression. A common method is to extract RNA from cells, then isolate the mRNA from other types of RNA, like ribosomal RNA or transfer RNA, by running the sample over a column of beads with stretches of thymine nucleotides attached.

These bind to the poly-A tail, a chain of adenine nucleotides specifically present on the 3-prime ends of eukaryotic mRNA. The other types of RNA do not bind and are washed away.

After the mRNA is isolated, a poly-T primer is bound to the poly-A tail, providing a starting point for reverse transcriptase enzymes to transcribe a single-stranded cDNA from the mRNA. Chemicals, such as RNase enzymes, are then added to degrade the RNA.

DNA polymerase enzymes are then used to synthesize a strand complementary to the cDNA, resulting in double-stranded cDNA, which can be inserted into a bacterial or viral vector and used in molecular biology research.

15.13: Complementary DNA

Overview

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA&mdashunlike genomic DNA&mdashalso allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene.

CDNA Synthesis

cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types&mdashtransfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail&mdasha string of adenine nucleotides&mdashadded to its 3&rsquo end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away.

Next, reverse transcriptase&mdasha DNA polymerase enzyme from retroviruses&mdashis used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3&rsquo end of a chain, a poly(T) primer is added to bind to the poly(A) tail to provide a starting point for cDNA synthesis. The cDNA strand ends in a hairpin loop. The RNA is then degraded&mdashcommonly with alkali treatment or RNase enzymes&mdashleaving the single-stranded cDNA intact.

A second DNA strand complementary to the cDNA is then synthesized by DNA polymerase&mdashoften using the hairpin loop of the first cDNA strand or a nicked piece of the mRNA as a primer.

The resulting double-stranded cDNA can be inserted into bacterial or viral vectors and cloned using standard molecular biology techniques. A cDNA library&mdashrepresenting all the mRNAs in the cells or tissue of interest&mdashcan also be constructed for additional research.

Pray, Leslie A. &ldquoThe Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes.&rdquo Nature Education 1, no. 1 (2008): 94. [Source]


Watch the video: cDNA Complementary (January 2022).