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58 terms

Recombinant DNA Technology and Genomics

Chapter 3
STUDY
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Genetic engineering
The process of altering an organism′s DNA. This is usually by design.
Restriction Enzymes
DNA-cutting proteins found primarily in bacteria. Enzymes cleave (cut) the phosphodiester backbone of double-stranded DNA at specific nucleotide sequences (restriction sites). Commercially available restriction enzymes are essential for molecular biology experiments.
Plasmid DNA
Small, circular, self-replicating double-stranded DNA molecules found primarily in bacterial cells. Plasmids often contain genes coding for antibiotic resistance proteins and are routinely used for DNA-cloning experiments.
Substrates
Molecule or molecules on which an enzyme performs a reaction.
Palindromes
A word or phrase that reads the same forward and backward (for example, "a toyota"). In the context of biotechnology, a DNA sequence with complementary strands that reads the same forward and backward. Most restriction enzyme recognition sequences are palindromes.
Recognition sequence
Specific sequence of DNA nucleotides recognized and cut by a restriction enzyme.
Restriction Site
Specific sequence of DNA nucleotides recognized and cut by a restriction enzyme.
Cohesive ends
Overhanging single-stranded ends of a DNA molecule created by the action of certain restriction enzymes.
Blunt ends
Double-stranded ends of a DNA molecule created by the action of certain restriction enzymes.
Vectors
DNA (or viruses) that can be used to carry and replicate other pieces of DNA in molecular biology experiments; for example, plasmid DNA, viruses used for gene therapy; also refers to organisms that carry disease.
Transformation
The process by which bacteria take in DNA from the surroundings. Term also is used to define changes that cause a normal cell to become a cancer cell.
Electroporation
A process for transforming bacteria with DNA that uses electrical shock to move DNA into cells; can also be used to introduce DNA into animal and plant cells.
Antibiotic selection
Laboratory technique used to identify bacteria containing recombinant DNA of interest; involves growing bacteria on media with antibiotic or other selection molecules.
Origin of replication
Specific locations in a DNA molecule where DNA replication begins.
RNA polymerase promoter
Copies RNA from a DNA template; different forms of RNA polymerase synthesize different types of RNA.
Copy number
The number of copies of a particular DNA molecule or gene sequence such as a plasmid in a bacterial cell.
Insulin
Protein hormone produced by cells of the pancreas; involved in glucose metabolism by cells; deficiencies in insulin production or insulin-receptor production can cause different forms of diabetes.
Humulin
The recombinant form of human insulin, became the first recombinant DNA product to be approved for human applications by the U.S. Food and Drug Administration.
Lytic cycle
A process of bacteriophage replication that involves phages infecting bacterial cells and then replicating and rupturing (lysing) the bacterial host cells.
Plaques
Small clear spots of dead bacteria appearing on a culture plate, caused by bacterial cell lysis by bacteriophage.
Expression vectors
DNA vector such as a plasmid that can be used to produce (express) proteins in a cell.
Cosmid vectors
Large circular double-stranded DNA vector that is used for gene-cloning experiment in bacteriophages.
BAC
Large circular vectors that can replicate very large pieces of DNA; used to clone pieces of human chromosomes for the Human Genome Project.
YAC
Plasmid vectors grown in yeast cells that can replicate very large pieces of DNA; used to clone pieces of human chromosomes for the Human Genome Project.
Ti vectors
Plasmid DNA vector derived from soil bacterium that can be used to clone genes in plant cells and deliver genes into plants.
Complementary DNA libraries
DNA copies of all mRNA molecules expressed in an organism′s cells; can be "screened" to isolate genes of interest.
Reverse transcriptase
Viral polymerase enzyme that copies RNA into single-stranded DNA. This commercially available enzyme is used for many molecular biology experiments, such as creating cDNA.
Retroviruses
Viruses that contain an RNA genome and use reverse transcriptase to copy RNA into DNA during the replication cycle in host cells.
DNA probe
A single-stranded radioactive or nonradioactive DNA fragment that is complementary to the gene of interest
Oligonucleotide
Short, single-stranded synthetic DNA sequences; used in PCR reactions and as DNA probes.
PCR
Laboratory technique for amplifying and cloning DNA; involves multiple cycles of denaturation, primer hybridization, and DNA polymerase synthesis of new strands.
Taq DNA polymerase
DNA-synthesizing enzyme isolated from Thermus aquaticus, a thermophilic Archae that lives in hot springs; its ability to withstand high temperatures (thermostable) without denaturation makes it valuable for use in PCR experiments.
Restriction map
An arrangement or "map" of the number, order, and types of restriction-enzyme cutting sites in a DNA molecule.
Agarose gel electrophoresis
Laboratory procedure that involves using an electrical charge to move and separate biomolecules of different sizes, such as DNA, RNA, and proteins, through a semisolid separating gel matrix. Examples include agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE).
Ethidium bromide
Tracking dye that penetrates (intercalates) between the base pairs of DNA. Commonly used to stain DNA in gels because ethidium bromide fluoresces when exposed to ultraviolet light.
DNA sequencing
Laboratory technique for determining the nucleotide "sequence" or arrangement of A, G, T, and C nucleotides in a segment of DNA.
Dideoxyribonucleotide
modified nucleotide. A ddNTP differs from a normal deoxyribonucleotide (dNTP) because it has a hydrogen group attached to the 3′ carbon of the deoxyribose sugar instead of a hydroxyl group-OH.
Fluorescence in situ hybridization
Laboratory technique that uses single-stranded DNA or RNA probes labeled with fluorescent nucleotides to identify gene sequences in a chromosome or cell in situ (Latin for "in its original place").
Southern blotting
Laboratory technique invented by Ed Southern that involves transferring (blotting) DNA fragments onto a filter-paper blot for use in probe hybridization studies.
Northern blotting
Laboratory technique for separating RNA molecules by gel electrophoresis and transferring (blotting) RNA onto a filter paper blot for use in hybridization studies.
Western blotting
Laboratory technique for separating protein molecules by gel electrophoresis and transferring (blotting) proteins onto a filter paper blot that is usually probed with antibodies to study protein structure and function.
DNA microarray
A chip consisting of a glass microscope slide containing thousands of pieces of single-stranded DNA molecules attached to specific spots on the slide; each spot of DNA is a unique sequence.
Quantitative PCR
Uses primers made with fluorescent dyes and specialized thermal cyclers that enable researchers to quantify amplification reactions as they occur
Site-directed mutagenesis
With this technique, mutations can be created in specific nucleotides of a cloned gene contained in a vector. The gene can then be expressed in cells, which results in translation of a mutated protein. This allows researchers to study the effects of particular mutations on protein structure and functions as a way to determine what nucleotides are important for specific functions of the protein.
siRNA
Small (21 or 22 nt) double-stranded pieces of nonprotein coding RNA, so named because they were shown to bind to mRNA and subsequently block or interfere with translation of bound mRNAs.
genomics
The study of genomes.
bioinformatics
Interdisciplinary science that involves developing and applying information technology (computer hardware and software) for analyzing biological data such as DNA and protein sequences; also includes the use of computers for the analysis of molecular structures and creating databases for storing and sharing biological data.
national center for biotechnology information
formed the RAC which was charged with evaluating risks of recombinant DNA technology
GenBank
Renowned public database of DNA sequences provided by researchers throughout the world; resources for sharing and analyzing DNA sequence information.
Semiconservative replication
Process by which DNA is copied; one original (parent) DNA molecule gives rise to two molecules, each of which has one original strand and one new strand.
Helicase
Enzyme that separates two strands of a DNA molecule during DNA replication.
Single-stranded binding protein
Proteins that bind to unraveled single strands of parental DNA during DNA replication to prevent DNA from reforming double strands before being copied.
Origin of replication
Specific locations in a DNA molecule where DNA replication begins.
Primase
Oligonucleotides complementary to specific sequences of interest; used in PCR reactions to amplify DNA and DNA-sequencing reactions.
DNA polymerase
Key enzyme that copies strands of DNA during DNA replication to create new strands of nucleotides; has important applications for synthesizing DNA in molecular biology experiments.
Leading Strand
During DNA replication, the strand of newly synthesized DNA that is copied by DNA polymerase in a continuous fashion, 5′ to 3′ into the replication fork.
Lagging Strand
During DNA replication, the strand of newly synthesized DNA that is copied by DNA polymerase in a discontinuous (interrupted) fashion, 5′ to 3′ away from the replication fork as a series of short DNA pieces called Okazaki fragments.
DNA ligase
Enzyme that forms covalent bonds between incomplete (Okazaki) fragments of DNA during DNA replication; routinely used for joining DNA fragments in recombinant DNA experiments.