Chapter 5. Exploring Genes and Genomes Flashcards
An artificial bacterial chromosome, a highly engineered version of the E. coli fertility (F factor), that can serve as a cloning vector for inserts as larges as 300 kb.
Bacterial artificial chromosome (BAC)
A means of introducing a variety of mutations into a gene of interest. A short segment of plasmid harboring the original gene is removed by restriction enzyme treatment. A synthetic double-stranded oligonucleotide (the cassette) carrying the genetic alterations of interest is subsequently inserted.
cassette mutagenesis
Endonuclease enzymes that recognize specific base sequences in double-stranded DNA and cleave both strands of the duplex at specific places.
Restriction enzyme
a word, sentence, or verse that reads the same from right to left as it does from left to right. An example is radar. By extension to biochemistry, a sequence of double-stranded DNA that is the same in each strand when the strands are read in the same direction; that is, it displays a twofold rotational symmetry, for example: 3inches-CCTAGG-5inches/5inches-GGATCC-3 inches.
Palindrome
DNA complementary to an mRNA sequence.
Complementary DNA (cDNA)
a collection of DNA fragments, inserted into vector molecules, that represents the entire genome of an organism.
Genomic library
A class of mobile genetic elements that can be up to 10 kbp in length that appear more than a million times in the human genome.
long interspersed elements (LINES)
a technique analogous to Southern blotting, in which a mixture of RNA fragments is separated by electrophoresis, transferred to a nitrocellulose sheet, hybridized to a radioactively labeled DNA probe complementary to the desired sequence, and visualized by autoradiography. The technique can therefore be used to locate and identify an RNA fragment containing a specific sequence.
Northern blotting
A bacteriophage cloning vector that can be incorporated into the hosts genome and thus be replicated indefinitely or can be expressed and destroy the host.
Lambda (λ) phage
an enzyme that catalyzes the formation of a phosphodiester bond between the 3 inch-OH at the end of one DNA chain and the 5 inch-phosphate at the end of the other chain; it is involved in the synthesis, repair, and splicing of DNA.
DNA ligase
A plasmid-cloning vector that has been optimized for the expression of large amounts of recombinant protein encoded by the vector.
Expression vector
A DNA-sequencing technique that employs controlled interruption of enzymatic replication of the molecule to be analyzed. DNA polymerase I is used with a primer, the four deoxynucleoside triphosphates, and a 2’,3’-dideoxy analog of one of them. Fragments of various lengths are produced in which the dideoxy analog is at the 3’ end. Four sets of chain-terminated fragments (one for each analog) are then displayed by electrophoresis and autoradiography, and the base sequence can be read from the four lanes of the gel.
Controlled termination of replication (Sanger dideoxy method)
A means of transforming plant cells. DNA is coated onto 1-mm-diameter tungsten pellets, and these microprojectiles are fired at the target cells with a velocity greater than 400 m s�1.
Gene gun (bombardment-mediated transformation)
Highly specific modification of genomic DNA.
genome editing
Inactivating a gene and looking for resulting abnormalities in order to determine the gene’s function.
gene disruption (gene knockout)
A collection of all of the complementary DNA for all mRNA that a cell contains, which have been inserted into vectors, and then inserted into bacteria.
cDNA library
A solid support such as a microscope slide to which are affixed oligonucleotides or cDNAs corresponding to specific genes. Fluorescently labeled cDNA is hybridized to the slide to reveal the expression level for each gene, identifiable by its known position within the microarray.
DNA microarray (gene chip)
These are plasmids or bacteriophage that allow the insertion and replication of DNA fragments into bacteria for the purpose of cloning. They often feature a polylinker region that includes many unique restriction sites within its sequence, allowing the region to be cleaved with many different restriction enzymes or combinations of enzymes. This provides great versatility in the DNA fragments that can be inserted.
cloning vectors
Small, mobile, non-coding DNA sequences, related to transposons, that can replicate and insert at random sites in the genome.
mobile genetic element
a technique used to locate and identify a DNA fragment containing a specific sequence; a mixture of fragments is separated by electrophoresis, transferred to a nitrocellulose sheet, hybridized to a radioactively labeled DNA probe complementary to the desired sequence, and visualized by autoradiography.
Southern blotting
a DNA molecule that can be used to clone DNA inserts ranging from 100 to 1000 kb in length; these molecules contain a centromere, an autonomously replicating sequence, a pair of telomeres, selectable marker genes, and an insertion site for the sequence to be cloned.
Yeast artificial chromosome (YAC)
Circular duplex DNA molecules that replicate autonomously and act as accessory chromosomes in bacteria; they carry useful genes but are disposable under certain conditions.
Plasmid
Complementary single-stranded ends, also called cohesive ends, produced by restriction enzymes that cleave their palindromic target sequences in a staggered fashion.
Sticky ends
A DNA molecule that can replicate autonomously in an appropriate host organism. Vectors are designed to enable the rapid, covalent insertion of DNA fragments of interest.
Vector
