AP BIO CH 20 DNA Technology and Genomics

using restriction fragment analysis

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nucleic acid

A polymer (polynucleotide) consisting of many nucleotide monomers; serves as a blueprint for proteins and, through the actions of proteins, for all cellular activities. the two types are DNA and RNA

genomic library

A set of thousands of DNA segments from a genome, each carried by a plasmid, phage, or other cloning vector.

sticky end

A single-stranded end of a double-stranded DNA restriction fragment.

restriction site

A specific sequence on a DNA strand that is recognized as a "cut site" by a restriction enzyme

Human Genome Project

An international collaborative effort to map and sequence the DNA of the entire human genome.

genetic engineering

The direct manipulation of genes for practical purposes.

southern blotting of DNA fragments

researchers can detect specific nucleotide sequences within a DNA sample with this method. In particular, Southern blotting is useful for comparing the restriction fragments produced from different sample of genomic DNA.

nucleic acid probe hybridization

1) A special filter paper is pressed agianst the master plate, transferring cells to the bottom side of the filter. Marks (Xs) are placed on the filter paper and agar to establish the position of each individual colony in reference to the marks. 2) The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. Radioactive probe molecules complementary to part of the gene of interest are incubated with the filter. The single-stranded probe base-pairs with any complementary DNA on the filter; excess DNA is rinsed off. 3) The filter is laid under photographic film, allowing any radioactive areas to expose the film. Black spots on the film corresond to the locations on the filter of DNA that has hybridized to the probe. 4) After the developed film is flipped over, the reference marks on the on the film and master plate are aligned, so that colonies carrying the gene of interest can be located.

clone

1) a lineage of genetically identical individuals or cells. (2) in popular usage, a single individual organism that is genetically identical to another individual (3) As a verb, to make one or more genetic replicas of an individual or cell

overview of gene cloning

1) gene inserted into plasmid 2) plasmid put into bacterial cell 3) host cell grown in culture to form a clone of cells containing the "cloned" gene of interest 4) basic research and various applications

cloning a human gene in a bacterial plasmid

1) isolate plasmid DNA from bacterial cells and DNA from human cells containing the gene of interest 2) Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA 3) Mix the cut plasmids and DNA fragments. Some join by base pairing; add DNA ligase to seal them together. The products are recombinant plasmids. 4) Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Under suitable conditions, some cells will take up a recombinant plasmid or other DNA molecule by transformation. 5) Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow.

restriction enzyme

1) restriction enzyme cuts the sugar phosphate backbones at each arrow 2) DNA fragment from another source is added. Base pairing of sticky ends produces various combinations 3) DNA ligase seals the strands

expression vector

A cloning vector that contains the requisite prokaryotic promoter just upstream of a restriction site where a eukaryotic gene can be inserted.

restriction enzyme

A degradative enzyme that recognizes and cuts up DNA (including that of certain phages) that is foreign to a bacterium.

linkage map

A genetic map based on the frequencies of recombination between markers during crossing over of homologous chromosomes.

physical map

A genetic map in which the actual physical distances between genes or other genetic markers are expressed, usually as the number of base pairs along the DNA

Southern blotting

A hybridization technique that enables researchers to determine the presence of certain nucleotide sequences in a sample of DNA.

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 39 end of a new DNA fragment to the 59 end of a growing chain.

electroporation

A technique to introduce recombinant DNA into cells by applying a brief electrical pulse to a solution containing cells. The electricity creates temporary holes in the cells' plasma membranes, through which DNA can enter.

yeast artificial chromosomes (YAC)

A vector that combines the essentials of a eukaryotic chromosome-an orgin for DNA replication, a centromere, and two telomeres- with foreign DNA

Cloning vector

An agent used to transfer DNA in genetic engineering. A plasmid that moves recombinant DNA from a test tube back into a cell is an example of a cloning vector, as is a virus that transfers recombinant DNA by infection.

Nucleic Acid Probe Hybridization (Result)

Colonies of cells containing the gene of interest have been identified by nucleic acid hybridization. Cells from colonies tagged with the probe can be grown in large tanks of liquid growth medium. large amounts of the DNA containing the gene of interest can be isolated from these cultures. By using probes with different nucleotide sequences, the collection of bacterial clones can be screened for different genes.

restriction fragment

DNA segment resulting from cutting of DNA by a restriction enzyme

restriction fragment length polymorphisms (RELPs)

Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths (DNA segments resulting from treatment with restriction enzymes); useful as genetic markers for making linkage maps

denaturation

For proteins, a process in which a protein unravels and loses its native conformation, thereby becoming biologically inactive. For DNA, the separation of the two strands of the double helix. Denaturation occurs under extreme conditions of pH, salt concentration, and temperature.

nucleic acid probe

In DNA technology, a labeled single-stranded nucleic acid molecule used to tag a specific nucleotide sequence in a nucleic acid sample. Molecules of the probe hydrogen-bond to the complementary sequence wherever it occurs; radioactive or other labeling of the probe allows its location to be detected.

hybridization

In genetics, the mating, or crossing of two true-breeding varieties

biotechnology

The manipulation of living organisms or their components to produce useful products.

gene cloning

The production of multiple copies of a gene

gel electrophoresis

The separation of nucleic acids or proteins, on the basis of their size and electrical charge, by measuring their rate of movement through an electrical field in a gel.

Recombinant DNA

a DNA molecule made in vitro with segments from different sources.

cDNA library

collection of clones containing DNA made from a mRNA molecule

DNA sequencing

the process of determining the order of nucleotides in a DNA molecule