S1 End Mapping:
A technique to determine where the end of an RNA transcript lies with respect to its template DNA (the gene).
A fluorescence detection system consists of a detection probe with the upstream primer with a fluorophore at the 5' end, followed by a complementary stem-loop structure also containing the specific probe sequence, quencher dye and a PCR primer on the 3' end. Between the primer and its tail (the probe), a blocking agent (DNA spacer, hexaethylene glycol) is placed. This structure makes the sequence-specific priming and probing a unimolecular event that creates enough specificity for allelic discrimination assays.
To screen a library is to select and isolate individual clones out of the mixture of clones. For example, if you needed a cDNA clone of the pituitary glycoprotein hormone alpha subunit, you would need to make (or buy) a pituitary cDNA library, then screen that library in order to detect and isolate those few bacteria carrying alpha subunit cDNA.
There are two methods of screening which are particularly worth describing: screening by hybridization, and screening by antibody.
Screening by hybridization involves spreading the mixture of bacteria out on a dozen or so agar plates to grow several ten thousand isolated colonies. Membranes are laid onto each plate, and some of the bacteria from each colony stick, producing replicas of each colony in their original growth position. The membranes are lifted and the adherent bacteria are lysed, then hybridized to a radioactive piece of alpha DNA. When X-ray film is laid on the filter, only colonies carrying alpha sequences will "light up". Their position on the membranes show where they grew on the original plates, so you now can go back to the original plate (where the remnants of the colonies are still alive), pick the colony off the plate and grow it up.
Screening by antibody is an option if the bacteria and plasmid are designed to express proteins from the cDNA inserts.The principle is similar to hybridization, in that you lift replica filters from bacterial plates, but then you use the antibody (perhaps generated after olde tyme protein purification rituals) to show which colony expresses the desired protein.
Although in most cases sense DNA refers to the DNA strand whose sequences code for proteins, in some cases sense/antisense (e.g., for PCR primers) are arbitrarily used to distinguish the two strands of the double-stranded DNA. RNA (sense strand) is transcribed from the antisense DNA strand.
Similarities in nucleic acid sequence and organization, and in their encoded products, that are sufficiently great as to imply common ancestral origins.
Similarity in nucleic acid or polypeptide sequences, particularly in shorter segments, that may not be sufficient to imply common ancestral origins.
Determination of the order of nucleotides (base sequences) in a DNA or RNA molecule or the order of amino acids in a protein.
A consensus sequence (AGGAGG) upstream of the translation initiation site ATG in bacteria. Important for translation initiation.
Short Arm (p):
One of the two prominent segments of a chromosome; the long or "q" arm is the other. The arms of a given chromosome join at its centromere.
The practice of randomly clipping a larger DNA fragment into various smaller pieces, cloning everything, and then studying the resulting individual clones to figure out what happened. For example, if one was studying a 50 kb gene, it "may" be a bit difficult to figure out the restriction map. By randomly breaking it into smaller fragments and mapping those, a master restriction map could be deduced.
A way of determining the sequence of a large DNA fragment which requires little brainpower but lots of late nights. The large fragment is shotgun cloned (see above), and then each of the resulting smaller clones ("subclones") is sequenced. By finding out where the subclones overlap, the sequence of the larger piece becomes apparent. Note that some of the regions will get sequenced several times just by chance.
A type of cloning vector that contains sequences enabling it to be propagated and maintained in more than one type of host (e.g. E. coli and mammalian cells). For this purpose shuttle vectors carry different origin of DNA replication which are characteristic of the desired host systems.
The introduction of a mutation, usually a point mutation or an insertion, into a particular location in a cloned DNA fragment. This mutated fragment may be used to "knock out" a gene in the organism of interest by homologous recombination.
Occurs between two specific but not necessarily homologous sequences. Usually catalyzed by enzymes not involved in general or homologous recombination.
Subunit of bacterial RNA polymerase which controls the correct initiation of transcription. These proteins increase the binding affinity of RNA polymerase to a promoter. Different sigma factors recognize different promoter sequences.
Signal Recognition Particle (SRP):
A chaperonin complex responsible for arresting polypeptide synthesis and facilitating the docking of a ribosome to the endoplasmic reticulum membrane. Normally, on ribosomes translating polypeptides destined for insertion into or across the endoplasmic reticulum membrane become associated with SRP.
An enzyme present within the lumen of the endoplasmic reticulum which proteolytically cleaves a secreted protein at the site of a signal sequence.
A hydrophobic amino acid sequence which directs a growing peptide chain to be secreted into the endoplasmic reticulum.
A nucleotide substitution (never a single deletion or insertion) which does not alter the amino acid sequence of an encoded protein due to the degeneracy of the genetic code. Such mutations usually involve the third base (wobble position) of codons.
Simple Sequence Repeat (SSR):
Short, tandem repeats that are useful as genetic markers.
Hereditary disorder caused by a mutant allele of a single gene (e.g., Duchenne muscular dystrophy, retinoblastoma, sickle cell disease). Compare polygenic disorders.
Single Nucleotide Polymorphism (SNP): Single base differences of DNA sequences between individuals of a population.
Single-Strand Conformational Polymorphism (SSCP):
Relies on secondary and tertiary structural differences between denatured and rapidly cooled amplified DNA fragments that differ slightly in their DNA sequence; different SSCP alleles are resolved on non-denaturing acrylamide gels, usually at low temperature; ability to resolve alleles depends on conditions of electrophoresis.
Small Inhibitory RNA that targets RNA degradation or inhibits translation of target genes.
Slope: Mathematically calculated slope of standard curve, e.g., the plot of Ct values against logarithm of ten-fold dilutions of target nucleic acid.
The analyte is put onto the membrane using a slot-shaped template. The template produces a consistently shaped spot, thus decreasing errors and improving the accuracy of the analysis.
Small Nuclear Ribonucleoprotein Particle (snRNP): Complexes formed by snRNA and proteins. Involved in RNA processing such as splicing and polyadenylation.
Small Nuclear RNA (snRNA):
Short RNAs, and forms complexes with proteins in snRNPs. snRNAs are involved in RNA processing such as splicing and polyadenylation.
Small nuclear RNA; forms complexes with proteins to form snRNPs; involved in RNA splicing, polyadenylation reactions, other unknown functions.
snRNP: "snerps", Small Nuclear RiboNucleoProtein particles, which are complexes between small nuclear RNAs and proteins, and which are involved in RNA splicing and polyadenylation reactions.
Single Nucleotide Polymorphism (SNP) - a position in a genomic DNA sequence that varies from one individual to another. It is thought that the primary source of genetic difference between any two humans is due to the presence of single nucleotide polymorphisms in their DNA. Furthermore, these SNPs can be extremely useful in genetic mapping (see 'Genetic Mapping') to follow inheritance of specific segments of DNA in a lineage. SNP-typing is the process of determining the exact nucleotide at positions known to be polymorphic.
A method closely related to RNase protection. Solution hybridization is designed to measure the levels of a specific mRNA species in a complex population of RNA. An excess of radioactive probe is allowed to hybridize to the RNA, then single-strand specific nuclease is used to destroy the remaining unhybridized probe and RNA. The "protected" probe is separated from the degraded fragments, and the amount of radioactivity in it is proportional to the amount of mRNA in the sample which was capable of hybridization. This can be a very sensitive detection method.
Refers to non-germline cells. Somatic cells may become terminally differentiated with alterations in their overall genetic compliment, because they are not responsible for passing along the organism's genetic material to the offspring.
Any cell in the body except gametes and their precursors.
A very high frequency of mutational events that occur in specific loci, such as the variable segments of expressed immunoglobulin genes. Somatic hypermutation in immunoglobulin genes occurs after all rearrangements have occurred and provide additional potential for variation in immunoglobulin structure.
Southern blot: A technique for detecting specific DNA using a labeled DNA probe. DNA (e.g. genomic DNA) is separated by electrophoresis in agarose gel and transferred onto a membrane. The membrane is then probed with labeled DNA to detect the specific sequence. Named after its inventor, Dr. Southern.
The binding of protein to a nucleic acid on a matrix similar to what is done for western, northern, and southern blots. This technique is used to identify DNA binding proteins and the recognition sites for these proteins.
A sequence within an mRNA molecule that is the site at which splicing occurs.
The process that removes introns (non-protein-coding portions) from transcribed RNAs. Exons (protein-coding portions) can also be removed. Depending on which exons are removed, different proteins can be made from the same initial RNA or gene. Different proteins created in this way are 'splice variants' or 'alternatively spliced'.
Catalyzes the splicing of precursor message RNA. Major components are snRNPs (U1, U2, U4, U5 and U6).
SP6 RNA Polymerase:
A bacteriophage RNA polymerase which is commonly used to transcribe plasmid DNA into RNA. The plasmid must contain an SP6 promoter upstream of the relevant sequence.
Simple Sequence Repeat.
Plasmid or retrovirus vectors are stably integrated into the genome of tissue culture cells, resulting in the stable expression of transfected genes. Clones of stably transfected cells are selected by selectable markers (e.g., G418 resistant gene) on the vectors.
A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, a standard curve is created from which the quantity of an unknown sample can be calculated.
Obtained by plotting Ct values against log-transformed concentrations of serial ten-fold dilutions of the target nucleic acid. Standard curve is obtained for quantitative PCR and the range of concentrations included should cover the expected unknown concentrations range. It is used to find out the dynamic range of the target (and/or normalizer), to calculate the slope (therefore, efficiency), r and R2 coefficients and also to help with quantitation.
Digestions of double-stranded DNA by many restriction enzymes (e.g. EcoR I) generate ends with a short single-stranded sequences. Such ends are called sticky ends.
Hybridization conditions: higher the stringency, lower the probability of hybridization. Increase the temperature or decreasing the salt concentration raises the stringency.
Cloning of the original DNA in a vector to other vectors. The original DNA may be digested to create smaller subclones.
A DNA sequence element to which transcriptional factors bind. Binding of transcriptional factors decreases gene transcription.
SYBR® Green I:
A fluorogenic minor groove binding dye that emits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA. It is used as a cheaper alternative in real-time PCR applications. It does not bind to ssDNA but because of the lack of sequence specificity it binds to any dsDNA product. Its use usually requires melting curve analysis to assure specificity of the results.