ch 20 molecular technologies Flashcards
recombinant dna technology
the use of in vitro molecular techniques to to manipulate fragments of dna to produce new arrangements
recombinant dna molecules
molecules that are produced in a test tube by covalently linking dna fragments from two different sources
gene cloning
the production of many copies of a gene using molecular methods, such as pcr or the introduction of the gene in to a vector that replicates in a host cell
vector
a small dna molecule that is used as a carrier of a dna segment into a cloning experiment
host cell
a cell that is infected with a virus of bacterium, or one that harbors a vector
plasmid
a general term for a dna molecule (most often circular) that exists independently of the chromosomal dna; some plasmids are used as vectors in cloning experiments
R factor
a type of plasmid found commonly in bacteria that gives resistance to a toxic substance, such as an antibiotic
origin of replication
a site on a chromosome that functions as an initiation site for the assembly of several proteins that begins the process of dna replication
selectable marker
a gene that provides a selectable phenotype in a cloning experiment; many markers are genes that give antibiotic resistance
shuttle vector
a cloning vector that can propagate in two or more different species, such as e. coli or yeast
expression vector
a cloning vector that contains a promoter so that the gene of interest is transcribed into rna when the vector is introduced into a host cell
restriction enzyme
(also called restriction endonuclease) an endonuclease that cleaves dna; restriction enzymes used in cloning experiments bind to specific base sequences and then cleave the dna backbone at two defined locations, one in each strand
dna ligase
an enzyme that catalyzes the formation of a covalent bond within the sugar-phosphate backbones of two dna strands
palindromic
describes sequences in the two strands of a dna molecule that are identical when read in opposite directions
recombinant vector
a vector that contains an inserted fragment of dna such as a gene from a chromosome
competent cells
cells that can take up dna from the environment or an extracellular medium
transformation
- when a bacterial cell takes up a plasmid vector or segments of chromosomal dna from the environment
- when a normal cell is converted into a malignant cell
reverse transcriptase
an enzyme that uses an rna template to make a double stranded dna molecule
complementary dna (cDNA)
dna that is made form an rna template by the action of reverse transcriptase
dna library
a collection of many recombinant vectors, each carrying a particular fragment od dna from a larger source; for example, each recombinant vector in a dna library might carry a small segment of chromosomal dna from a particular species
genomic library
a dna library of recombinant vectors that carry chromosomal dna fragments
cDNa library
a dna library whose recombinant vector carry cdna inserts
polymerase chain reaction (PCR)
the method for amplifying a dna region involving the sequential use of oligonucleotide primers and Taq polymerase
primer
a short segment of dna or rna that initiates dna replication
template dna
a sample of dna, such as chromosomal dna, that is used in a pcr experiment
Taq polymerase
a thermostable form of dna polymerase used in pcr experiments
primer annealing
in PCR, the process in which a primer binds to a strand of template dna
primer extension
in pcr, the step during which complementary strands of dna are synthesized from the denatured template dna, staring primers
thermocycler
a device that automates the timing of temperature changes in each cycle of a pcr experiment
reverse transcriptase PCR
a modification of pcr in which the first round of replication involves the use of rna and reverse transcriptase to make a complementary strand of dna
real-time pcr
a method of pcr in which the synthesis of dna is monitored in real time; this method can quantitate the starting amount of dna in a sample
cycle threshold method
(also called Ct method) in quantitative pcr, a method of determining the starting amount of dna based on a threshold level at which the accumulation of fluorescence in significantly greater than the background level
dna sequencing
a method for determining the base sequence in a segment of dna
dideoxy sequencing
a method of dna sequencing that uses dideoxyribonucleotides to terminate growth of dna strands
dideoxyribonucleotide (ddNTP)
a nucleotide used in dna sequencing that is missing the 3’ –OH group; if a dideoxyribonucleotide is incorporated into a dna strand, it stops any further growth of the strand
chain termination
an event that stops the growth of a dna stand, rna strand or polypeptide
automated dna sequencing
the use of fluorescently labeled dideoxyribonucleotides and a fluorescence detector to sequence dna
sequencing ladder
a series of bands on a gel that can be followed in order (ie from the bottom of the gel to the top of the gel) to determine the base sequence of a stand of dna
gene editing
experimentally altering the sequence of a gene
site-directed mutagenesis
a gene-editing technique that allows a researcher to produce a mutation at a specific location within a cloned dna segment by using an oligonucleotide primer with a mismatch
CRISPR-Cas technology
a method that uses the components of the crispr-cas system of genome defense found in prokaryotes to introduce mutations into genes
northern blotting
a technique used to detect a specific rna within a mixture of many rna molecules
western blotting
a technique used to detect a specific protein within a mixture of proteins
antibodies
(also known as immuniglobins) proteins that are produced by the immune system of vertebrates, which recognize foreign material (ie viruses, bacteria, etc) and target it for destruction
epitope
the structure on the surface of an antigen that is recognized by an antibody
antigen
foreign substances that elicit an immune response because they are recognized by antibodies
electrophoretic mobility shift assay (EMSA)
(also called gel retardation assay) a technique for studying protein-dna or protein-rna interactions in which the binding of proteins to dna fragments retards its mobility during gel electrophoresis
DNase I footprinting
a method for studying protein-dna interactions in which the binding a protein to dna protects the dna from digestion by dnase i
