In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing. Replication begins at a specific site in the DNA called the origin of replication. DNA replication is bidirectional from the origin of replication. To begin DNA replication, unwinding enzymes called DNA helicases cause the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks. These replication forks are the actual site of DNA copying. Helix destabilizing proteins bind to the single-stranded regions so the two strands do not rejoin. Enzymes called topoisimerases produce breaks in the DNA and then rejoin them in order to relieve the stress in the helical molecule during replication. As the strands continue to unwind and separate in both directions around the entire DNA molecule, the hydrogen bonding of free DNA nucleotides with those on each parent strand produces new complementary strands. As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. Actually, the nucleotides lining up by complementary base pairing are deoxynucleoside triphosphates, composed of a nitrogenous base, deoxyribose, and three phosphates. As the phosphodiester bond forms between the 5' phosphate group of the new nucleotide and the 3' OH of the last nucleotide in the DNA strand, two of the phosphates are removed providing energy for bonding. In the end, each parent strand serves as a template to synthesize a complementary copy of itself, resulting in the formation of two identical DNA molecules.
Transcription is the process through which a DNA sequence is enzymaticaly copied by an RNA polymerase to produce a complementary RNA. Or, in other words, the transfer of genetic information from DNA into RNA. In the case of protein-encoding DNA, transcription is the beginning of the process that ultimately leads to the translation of the genetic code (via the mRNA intermediate) into a functional peptide or protein. Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for DNA; therefore, transcription has a lower copying fidelity than DNA replication. Like DNA replication, transcription proceeds in the 5' to 3' direction (ie the old polymer is read in the 3' to 5' direction and the new, complementary fragments are generated in the 5' to 3' direction). Transcription is divided into 3 stages: initiation, elongation and termination.
Types of RNA:
- mRNA - messenger RNA is a copy of a gene. It acts as a photocopy of a gene by having a sequence complementary to one strand of the DNA and identical to the other strand. The mRNA acts as a busboy to carry the information stored in the DNA in the nucleus to the cytoplasm where the ribosomes can make it into protein.
- tRNA - transfer RNA is a small RNA that has a very specific secondary and tertiary structure such that it can bind an amino acid at one end, and mRNA at the other end. It acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mRNA.
- rRNA - ribosomal RNA is one of the structural components of the ribosome. It's sequence is the compliment of regions in the mRNA so that the ribosome can match with and bind to an mRNA it will make a protein from.
Hybridization on a microarray is similar to what occurs during other hybridization procedures, such as Southern blots or Northern blots. All these techniques rely on the complementary nature of nucleic acid bases. When two complementary strands of DNA or RNA are alongside each other, the bases match up with their complement, that is, thymine (or uracil) with adenine, and guanine with cytosine. On a DNA microarray, hybridization is affected by the same parameters as in these other techniques.
How the DNA is attached to the slide can affect how well the probe DNA and target DNA hybridize, especially for oligonucleotide microarrays (Fig. 8.24). The short length of oligonucleotides requires that the entire piece be accessible to hybridize. The length of the spacer between the oligonucleotides and the glass slide optimizes hybridization. An oligonucleotide attached with a short spacer has many of its initial nucleotides too close to the glass and inaccessible to incoming RNA or DNA. Oligonucleotides with longer spacers may fold back and tangle up. Oligonucleotides with medium-sized spacers are far enough from the glass, but not so far as to get tangled. Thus, medium-sized spacers give the best access for hybridization.
FIGURE 8.24. Length of Spacers and Target Molecules Affect Hybridization on Microarrays
(A) When the spacer between the glass slide and oligonucleotide is too short, the oligonucleotides are condensed and not accessible to hybridize. If the spacer region is too long, the oligonucleotides and spacers tangle and fold, preventing optimal hybridization. (B) When the target for hybridization is too long, the target sequences may form hairpins with themselves rather than bind to the array oligonucleotides.
Hybridization of two lengths of DNA (or RNA with DNA) depends on certain sequence features. One important property is the ratio of A:T base pairs to G:C base pairs. G:C base pairs have three hydrogen bonds holding them together, whereas A:T base pairs have only two hydrogen bonds. Thus, more GC base pairs give stronger hybridization. If the sequence has too many A:T base pairs, the duplex may form slowly and be less stable. Another important consideration is secondary structure. If the probe sequence can form a hairpin structure, it will hybridize poorly with the target. If the probe has several mismatches relative to the target, the duplex may not form efficiently. All these issues must be addressed when making an oligonucleotide microarray. Computer programs are available to identify suitable regions of genes with sequences that will produce effective probes.
cDNA arrays are less prone to the problems seen in oligonucleotide arrays. cDNAs are double-stranded, so secondary structures such as hairpins are less likely to be a problem. During a hybridization reaction, cDNA arrays must be denatured either with heat or chemicals, making the probes single-stranded. Then the single-stranded RNA samples are allowed to hybridize on the slide under conditions that promote duplex RNA:cDNA without any mismatches.
Oligonucleotide microarrays must have a sufficient spacer and little secondary structure in order to hybridize with the samples.
View chapterPurchase book
Read full chapter
URL: //www.sciencedirect.com/science/article/pii/B9780123850157000089
Molecular Biology
Jean L. Bolognia MD, in Dermatology, 2018
The Foundations of Molecular Techniques for Analyzing DNA, RNA, and Protein
The concepts behind molecular biology are simple and unifying. In general, they consist of extracting the molecules of interest, amplifying them to measurable amounts, and detecting them. Polymerase chain reaction (PCR) is a standard technique for amplifyingDNA (Table 3.1;Fig. 3.4)12. The PCR-amplified DNA, typically 50 to 2000 base pairs in size depending on the primers designed for a particular sequence, can be detected in a gel using an intercalating dye that fluoresces with ultraviolet light. The nucleotide sequence can then be determined via automated fluorescence sequencing techniques (Table 3.2;Fig. 3.5). This simple and relatively inexpensive approach is still widely used. However, it is being supplanted by massively parallel sequencing, also known as next-generation sequencing, in which millions of fragments of DNA are sequenced in a single run (seeTable 54.6)13.
RNA is also easy to purify, but it is much more readily degraded than DNA. Therefore, a typical first step in the analysis of RNA is to convert it into DNA using reverse transcription (RT;Table 3.3;Fig. 3.6A). Following RT, the complementary DNA (cDNA) can be amplified by PCR, as described above. The technique of RT-PCR has also been modified to allow accurate quantitation of very low levels of mRNA14. Because the amount of PCR product is monitored throughout each cycle of amplification, this technique is referred to as “real-time” quantitative PCR (Fig. 3.6B).
The amount ofprotein is a complex balance of synthesis and degradation controlled at multiple steps, including efficiency of protein translation and post-translational modifications that affect protein stability. One method used to measure levels of protein is referred to as a Western blot (Table 3.4;Fig. 3.7); it is also known as an immunoblot because an antibody is employed to detect the protein of interest. In addition to measuring protein levels, Western blot analysis can determine the size of proteins and can reveal whether there are different forms of the protein15. Another method commonly used to measure protein levels is an enzyme-linked immunosorbent assay (ELISA; seeTable 3.4)16. An ELISA can provide very exact quantitation of protein levels and may be less expensive and easier to perform than a Western blot.
View chapter on ClinicalKey
Chemical and Biochemical Approaches for the Study of Anesthetic Function, Part A
Paul Hoerbelt, Boris D. Heifets, in Methods in Enzymology, 2018
3.1.4 Notes
•
cDNA plasmids obtained from vendors or laboratories should be sequenced prior to use to ensure quality and sequence fidelity.
•For long-term storage (years), solubilized cDNA can be frozen at − 20°C in TE (Tris–EDTA) buffer at pH 8.0.
•For short-term storage (1 or 2 months), cDNA in our hands is stable in water or TE buffer at 4°C. The optimal concentration for storage is 1 mg/mL, which can be obtained if cDNA is first dissolved in water; TE buffer should not be concentrated.
•cDNA should never be vigorously vortexed, as this may cause shearing.
•Prior to PCR or transformation, it is preferable to have dilute DNA in water instead of TE buffer as EDTA may interfere with these reactions.
•Exposure to cDNA gel stains may be hazardous and appropriate precautions should be taken.
•UV glasses and UV face shield are essential for working with UV light.
•Long-wave UV light is preferable for extracting cDNA from gels, as it is less likely to cause damage to cDNA.
•PCR protocols may require substantial optimization before ideal products are obtained.
View chapterPurchase book
Read full chapter
URL: //www.sciencedirect.com/science/article/pii/S0076687918300259
Nucleic Acids and Molecular Genetics
Gerald Litwack Ph.D., in Human Biochemistry, 2018
Probing Libraries for Specific Genes
A cDNA probe can be generated from a specific mRNA. The mRNA, encoding a specific protein, is a template. By the action of reverse transcriptase and DNA polymerase, a cDNA is formed that can be used as a probe to hybridize with a specific gene sequence (Fig. 10.45).
Figure 10.45. Generation of a complementary DNA (cDNA) from an isolated eukaryotic gene.
Generally, the cDNA probe will be labeled, more recently, with a fluorescent tag that does not interfere with the hybridization reaction. Such a cDNA can be used to probe a library of cDNAs for a complementary sequence, either to find a longer sequence containing more information or to search out a full coding region of the gene.
View chapterPurchase book
Read full chapter
URL: //www.sciencedirect.com/science/article/pii/B9780123838643000107
Expression Arrays: Discovery and Validation
Neal M. Poulin, Torsten O. Nielsen, in Cell and Tissue Based Molecular Pathology, 2009
SPOTTED COMPLEMENTARY DNA MICROARRAYS
cDNA arrays are manufactured from the polymerase chain reaction (PCR) products of bacteriophage cDNA libraries, of which several suppliers maintain extensive collections available in microwell format. Collections are extensively curated and chosen to represent as many unique transcripts as possible, with preference to known transcripts and mapped positions but including uncharacterized and possibly chimeric sequences. cDNAs are long probes, 500 base pairs to 2 kilobases in length, and are generally 3′ biased because of the limitations of reverse transcription.
Probe clones are amplified by PCR within microwell plates, and the products are placed in a denaturing buffer and spotted by a simple robot. The robotics are designed to precisely position an array of micromachined capillary “pens.” Pens are dipped into the cDNA microwells, where they pick up a reproducible quantity of liquid. With submicron precision, the robot then repositions the pens at the appropriate point above the microarray and deposits cDNA by contacting the surface.
Although they have provided extraordinary insights and have enabled rapid progress, cDNA microarrays do have significant disadvantages. cDNA libraries are difficult to maintain, and cross-contamination of clones is common and almost inevitable. Multiple cross-infections and inviable plasmids are commonly observed, and each clone must be validated with every PCR run. Usually, a small fraction of suspect spots is associated with each print.
Another major problem with cDNA arrays is that probe affinities are widely variable across the array; thus, there may be numerous poorly performing probes. Probe–probe interactions can cause difficulties: probes are composed of double-stranded PCR products, so the antisense strand is present on the array and may contribute to probe–probe hybridizations. Further probe–probe interactions are ascribed to polyadenine–polythymine tracts, which are present in cDNA clones (because of oligo-deoxythymidine [oligo-dt] priming of reverse transcriptase). In addition, cDNA probes are of widely variable affinity because of uncontrolled variations in length and guanine–cytosine content. For these reasons, only relative measures of transcript abundance are practical with these arrays.
The specificity of hybridization to cDNA probes is also not ideal, and significant cross-hybridization has been shown among members of the same gene family. Depending on the specific sequence, this is estimated to be common for genes sharing more than 80% homology over the breadth of the probe.
Spotted cDNA microarrays have nonetheless served as reliable and reproducible hybridization platforms. This largely must be attributed to extensive curation of probe collections and efforts to empirically validate probe response. In particular, when there have been discordant results among array platforms, cDNA results have served as an indispensable reference source. They have also served as an important counterpoint to commercial platforms and have until recently been the only source of publicly available probe sequences.