Recombinant DNA
Review: These are DNA molecules that are formed from DNA segments from two different sources.
the manipulation of organisms or their components to make useful products
Define biotechnology.
the direct manipulation of genes for practical purposes
Define genetic engineering.
the bacterium Haemophilus influenzae
This was the first organism to have its entire genome sequenced in 1995.
plasmids
These are small circular DNA molecules that replicate separately from the bacterial chromosome.
gene cloning
This is the production of multiple copies of a single gene.
A bacterial plasmid is isolated so that foreign DNA can be inserted into it to create a recombinant DNA molecule. The plasmid is then returned to the bacterium, producing a recombinant bacterium. The divisions of the bacteria result in multiple copies of the gene and the protein expressed by the gene.
Describe, in detail, the process of gene cloning.
Restriction enzymes (restriction endonucleases)
Review: These enzymes can cut DNA molecules at a limited number of specific locations.
sugar-phosphate backbone
The restriction enzyme cuts the ______-______ _______ at each restriction site.
sticky end
The resulting double stranded fragment will have at least one single stranded end, called a ___________ ____.
DNA ligase
This is used to catalyze the formation of covalent bonds that close up the sugar-phosphate backbone between a fragment from a different DNA molecule used to fill the gap.
cloning vector
This is the DNA molecule that can carry foreign DNA into a host cell and replicate there.
bacterial plasmids
These are widely used as cloning vectors.
1) Isolate plasmid DNA from bacterial cells and DNA from hummingbird cells. The hummingbird DNA contains the gene of interest. 2) Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene (example of restriction site; another gene could have been chosen) and many cuts within the hummingbird DNA. 3) Mx the cut plasmids and DNA fragments. Some join by base pairing; add DNA ligase to seal them together. 4) Mix the DNA with bacterial cells that have a mutation in their own lacZ gene. 5) Plate the bacteria on agar containing ampicillin and X-gal (resembles lactose). Incubate until colonies grow.
Suppose you needed to create copies of a hummingbird gene using a bacterial plasmid as the clone vector. How would you proceed?
genomic library
The complete set of plsamid-containing cell clones, each carrying copies of a particular segment from the initial genome, is referred to as a ___________ __________.
bacterial artificial chromosome
These are large plasmids, trimmed down so that contain just the genes necessary to ensure replication and capable of carrying iserts of 100-300 kb.
1) Reverse transcriptase is added to a test tube containing mRNA isolated from the cell. 2) Reverse transcriptase makes the first DNA strand using the RNA as a template and a stretch of dTs (thymine bases to complement the poly-A-tail) as a primer. 3) mRNA is degraded by another enzyme. 4) A seconf DNA strand is created without introns (cDNA strand).
Describe the steps involved in the creation of a cDNA library for a eukaryotic gene.
nucleic acid hybridization
This is used to detect a gene based on a short, single stranded nucleic acid that can complementarily base-pair with the gene DNA.
nucleic acid probe
The strand of RNA or DNA used in nucleic acid hybridization is called a _____ _____ ____.
expression vector
This is a cloning vector that contains a highly actice bacterial promoted just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame.
yeast cells
These cells are used to clone eukaryotic genes to overcome the the incompatibility between eukaryotic cells and bacteria.
yeast artificial chromosome (YACs)
These combine the essentials of a eukaryotic chromosome – an origin for DNA replication, a centromere, and two telomeres – with foreign DNA.
electroporation
This is the process by which a brief electrical pulse is applied to a solution containing cells to create temporary holes in the plasma membranes, through which DNA can enter.
Polymerase chain reaction (PCR)
In this technique, any specific target segment within one or many DNA moleules can be quickly amplified in a test tube. A three-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
1) Denaturation: Heat breifly to separate the DNA strands. 2) Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence. 3) Extension: DNA pol III adds nucleotides to the 3′ end of each primer.
Describe the three cycles for one cycle in a polymerase chain reaction.
gel electrophoresis
This technique uses a gel that acts as a molecular seive to separate proteins based on size, electric charge, and other physical properties.
restriction fragment analysis
In this type of analysis, the DNA fragments produced by restriction enzyme digestion (cutting) of a DNA molecule are sorted by gel electrophoresis.
southern blotting
This technique combines gel electrophoresis with nucleic acid hybridization – allowing us to detect those bands with a certain gene.
Frederick Sanger
This researcher earned the 1980 Nobel prze for the development of dideoxy chain termination method.
nothern blotting
In this method, gel electrophoresis is carried out on mRNA samples from human embryos at different stages of development, transfer the samples to a nitrocellulose membrane, and then allow the mRNAs on the membrane to hybridize with a labeled probe recognizing the mRNA for the gene.
1) cDNA synthesis is carried out by incubating the mRNAs with reverse transcriptase and other necessary components 2) PCR amplification of the sample is performed using primers specific to the investigated gene 3) gel electrophoresis will reveal amplified amplified DNA products only in samples that contained mRNA transcribed from the investigated gene.
Describe the steps in reverse transcriptase – polymerase chain reaction (RT-PCR).
in situ hybridization
This method determines which tissues or cells are epxressing certian genes by tracking down the location of specific mRNAs using labeled probes in place, or in situ, in the itact organism.
DNA microarray assays
This consists of tiny amounts of a large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid.
in vitro mutagenesis
In this approach to determine gene function, specific mutations are introduced into a cloned gene, and then the mutated gene is introduced into a cloned gene, and then the mutated gene is returned to a cell in such a way that it disables the normla cellular copies of the same gene
totipotent
This describes the ability to dedifferentiate and then give rise to all the specialized cell types of the organism.
klon
Clone comes from this greek word meaning “twig”.
nuclear transplantation
This approach removes the nucleus of an unfertilized or fertilized egg and replace it with the nucleus of a differentiated cell.
The potential of a transplanted nucleus to direct normal development was inversely related to the age of the donor. In most animals, nuclear potential tends to be restricted more and more as embryonic development and cell differentiation progresses.
What Gurdon’s discovery about the potential of a transplanted nucleus to direct the normal development in the recipient cell?
by culturing mammary cells in nutrient-poor medium
How did the Scottish researchers who cloned Dolly acheive the necessary dedifferentiation of donor nuclei?
CC (Carbon Copy)
This cat was an example of nonidentity in clones.
stem cell
This is a relatively unspecialized cell that can both reproduce itself indefinitely and , under appropriate circumstances, differentiate into specialized cells of one or more types.
blastocyst stage
Stem cells can be isolated from human embryos at this stage.
embryonic stem (ES) cells
This the label given to cells taken from human embryos during the blastocyst stage.
adult stem cells
In contrast to ES cells, these stem cells are not able to give rise to all cell types in the organism.
pluripotent
This term describes an embryonic stem cell’s ability to give rise to a variety of different cell types
induced pluripotent stem (iPS) cells
These cells were transformed into ES cells via the introduction of four “stem cell” reglatory genes (brought about by clone copies made with retroviruses)
single nucleotide polymorphism (SNP)
A single base-pair site where variation is found in at least 1% of the population is called a ________ ___________ __________.
polymorphism
This is the general term for a variation in a DNA sequence.
once in 100 to 300 base pairs in the human genome and are found in both coding and noncoding DNA sequences
About how often do SNPs occur on average?
restriction fragment length polymorphism (RFLP)
Some SNPs alter the sequence recognized by a restriction enzyme and change the lengths of the restriction fragments formed by the digestion with that enzyme. This type of sequence change, which can occur in either coding or noncoding regions, is called a _________ _____ _____ ___________.
gene therapy
This is the introduction of genes into an afflicted individual for therapeutic purposes.
transgenic
Scientists can introduce a gene from an animal of one genotype into the genome of another individual. This individual is then called a ______ animal.
genetic profile
This is the term given to an individual’s unique set of genetic markers.
one chance in 10 billion and one in several trillion
In forensic cases using STR analysis with 13 markers, the probability of two people having identical DNA profiles is somewhere between ______________ (short answer).
Ti plasmid, from the soil bacterium Agrobacterium tumefaciens
This plasmid is the most common vector for introducing new genes into plant cells.
T DNA
The segment of the Ti plasmid that integrates itself into the genome of host cells is called _ ___.
