This technique is also used to produce labeled cDNA probes and is dependent on using random 6- to 10-base oligonucleotides to sit down on a single-stranded cDNA and then using DNA polymerase to synthesize the complementary strand using labeled nucleotides. This technique usually produces more favorable results than nick-translation.
The continuous collection of fluorescent signal from polymerase chain reaction throughout cycles.
Allele that determines phenotype only when homozygous; does not affect phenotype when heterozygous with a dominant allele.
Clones containing recombinant DNA molecules.
The combination of foreign DNA inserts with vector DNA (e.g., plasmid, phage, cosmid, etc.) to produce a clone within a host.
Recombinant DNA Technologies:
Procedures used to join together DNA segments in a cell- free system (an environment outside a cell or organism). Under appropriate conditions, a recombinant DNA molecule can enter a cell and replicate there, either autonomously or after it has become integrated into a cellular chromosome.
The process by which DNA is exchanged between pairs of equivalent chromosomes (crossing over) during egg and sperm formation. Recombination has the effect of making the chromosomes of the offspring distinct from those of the parents.
Total number of recombinants divided by the total number of progenies in a test cross.
A mode of filling a gap in one strand of duplex DNA by retrieving a homologous single strand from another duplex. Usually the underlying mechanism behind homologous recombination and gene conversion.
Enzymatic activity responsible for facilitating intra- or intermolecular recombination of DNA molecules.
A passive or active signal used to normalize experimental results. Endogenous and exogenous controls are examples of active references. Active reference means the signal is generated as the result of PCR amplification.
Used in all reactions to obtain normalized reporter signal (Rn) adjusted for well-to-well variations by the analysis software. The most common passive reference dye is ROX and is usually included in the master mix.
Regulatory Regions or Sequences:
A DNA base sequence that controls gene expression.
A surprising portion of any genome consists not of genes or structural elements, but of frequently repeated simple sequences. These may be short repeats just a few nt long, like CACACA etc. They can also range up to a few hundred nt long. Examples of the latter include Alu repeats, LINEs, SINEs. The function of these elements is often unknown. In shorter repeats like di- and tri-nucleotide repeats, the number of repeating units can occasionally change during evolution and descent. They are thus useful markers for familial relationships and have been used in paternity testing, forensic science and in the identification of human remains.
DNA replication unwinds a portion of the DNA helix, forming a fork like structure.
A segment of genomic DNA that contains an origin of replication and is replicated under the control of that origin.
Reporter Dye (Fluorophore):
The fluorescent dye used to monitor amplicon accumulation. This can be attached to a specific probe or can be a dsDNA-binding agent
The use of a functional enzyme, such as beta-galactosidase, luciferase, or chloramphenicol acetyltransderase, downstream of a gene, promoter, or translational control element of interest, to more easily identify successful introduction of the gene into a host and to measure transcription and/or translation.
A form of gene regulation wherein the promoter is prevented from assembling an RNA polymerase complex, so that transcription does not occur.
As applied to proteins, what remains of an amino acid after its incorporation into a peptide chain, with subsequent loss of a water molecule
Degree of molecular detail on a physical map of DNA, ranging from low to high.
It is a portion of a gene which must be present in order for that gene to respond to some hormone or other stimulus. Response elements are binding sites for transcription factors.
A class of enzymes ("restriction endonucleases") generally isolated from bacteria, which are able to recognize and cut specific sequences ("restriction sites") in DNA.
The piece of DNA released after restriction digestion of plasmids or genomic DNA.The term also describes the fragments detected on a genomic blot which carry the gene of interest.
A "cartoon" depiction of the locations within a stretch of known DNA where restriction enzymes will cut.
An enzyme which will make a DNA copy of an RNA template - a DNA-dependant RNA polymerase. RT is used to make cDNA; one begins by isolating polyadenylated mRNA, providing oligo-dT as a primer, and adding nucleotide triphosphates and RT to copy the RNA into cDNA.
Reverse Allele-Specific Hybridization:
This automated variant of allele-specific hybridization couples unlabeled synthetic oligonucleotides specific for a wild type or mutant sequence to a solid support that is then allowed to bind genomic sequences of the locus of interest, which have been amplified by PCR. The use of highly stringent conditions of hybridization allows differential binding of the amplified DNA to the wild type or mutant specific oligonucleotide and thereby allows genotypic determination of the individual.
Often, large families of homologous proteins exist and multiple previously unknown members of the family can be obtained by screening cDNA libraries under low stringency using cDNA or oligonucleotide probes from regions highly conserved amongst members of the family.
Restriction fragment length polymorphism; the acronym is pronounced "riflip". Although two individuals of the same species have almost identical genomes, they will always differ at a few nucleotides. Some of these differences will produce new restriction sites (or remove them), and thus the banding pattern seen on a genomic Southern will thus be affected. For any given probe (or gene), it is often possible to test different restriction enzymes until you find one which gives a pattern difference between two individuals - a RFLP. The less related the individuals, the more divergent their DNA sequences are and the more likely you are to find a RFLP.
A strand of RNA synthesized in-vitro (usually radiolabeled) and used as a probe for hybridization reactions. An RNA probe can be synthesized at very high specific activity, is single stranded (and therefore will not self anneal), and can be used for very sensitive detection of DNA or RNA.
A cellular particle which is involved in the translation of mRNAs to make proteins. Ribosomes are a complex consisting of ribosomal RNAs (rRNA) and several proteins.
A catalytically active RNA. A good example is the hepatitis delta virus RNA which is capable of self-cleavage and self-ligation in the absence of protein enzymes.
Ribosomal Binding Sequence (Shine-Dalgarno Sequence):
In prokaryotic organisms, part or all of the polypurine sequence AGGAGG located on mRNA just upstream of an AUG initiation codon; it is complementary to the sequence at the 3' end of 16S rRNA; and involved in binding of the ribosome to mRNA. The internal ribosomal entry site found in some viruses may be an analogous eukaryotic genetic element.
'RNA interference' (a.k.a. 'RNA silencing') is the mechanism by which small double-stranded RNAs can interfere with expression of any mRNA having a similar sequence. Those small RNAs are known as 'siRNA', for short interfering RNAs. The mode of action for siRNA appears to be via dissociation of its strands, hybridization to the target RNA, extension of those fragments by an RNA-dependent RNA polymerase, then fragmentation of the target. Importantly, the remnants of the target molecule appears to then act as an siRNA itself; thus the effect of a small amount of starting siRNA is effectively amplified and can have long-lasting effects on the recipient cell.
Ribonuclease; an enzyme which degrades RNA. It is ubiquitous in living organisms and is exceptionally stable. The prevention of RNase activity is the primary problem in handling RNA.
RNase Protection Assay:
This is a sensitive method to determine (1) the amount of a specific mRNA present in a complex mixture of mRNA and/or (2) the sizes of exons which comprise the mRNA of interest. A radioactive DNA or RNA probe (in excess) is allowed to hybridize with a sample of mRNA (for example, total mRNA isolated from tissue), after which the mixture is digested with single-strand specific nuclease. Only the probe which is hybridized to the specific mRNA will escape the nuclease treatment, and can be detected on a gel. The amount of radioactivity which was protected from nuclease is proportional to the amount of mRNA to which it hybridized. If the probe included both intron and exons, only the exons will be protected from nuclease and their sizes can be ascertained on the gel.
Enzymatic activity responsible for DNA-dependent synthesis of RNA. In prokaryotes there is only one RNA polymerase. In eukaryotes, there are three, each of which transcribes a different group of genes.
A complex and incompletly understood series of reactions occuring in the nucleus of eukaryotic cells in which pre-mRNA transcribed from chromosomal DNA is processed such that noncoding regions of the pre-mRNA (introns) are excised, and coding regions (exons) are covalently linked to produce an mRNA molecule ready for transport to the cytoplasm.
"ribosomal RNA"; any of several RNAs which become part of the ribosome, and thus are involved in translating mRNA and synthesizing proteins. They are the most abundant RNA in the cell
ROX: 6-carboxy-X-rhodamine. Most commonly used passive reference dye for normalization of reporter signal.
RT-PCR (Reverse Transcription PCR):
This technique allows the rapid amplification of cDNA starting with RNA. The first step of the reaction is to reverse-transcribe the RNA into a first strand cDNA copy using the enzyme reverse transcriptase. The primer for the reverse transcription can either be oligo dT, to hybridize to the polyadenylation tail, or the antisense primer that will be used in the subsequent PCR reaction. Following this first step, standard PCR is then performed to rapidly amplify large amounts of cDNA from the reverse transcribed RNA.