Biotechnology - answer✔✔refers to the use of living organisms to create products that are useful
to humans
Recombinant DNA - answer✔✔DNA from two or more sources...allows for creation of DNA
sequences not otherwise found in biological organisms
Basic Mechanism of gene cloning - answer✔✔1. isolate plasmid DNA from bacterial cell
2. isolate DNA of interest
3. Insert the gene of interest into plasmid DNA...forms recombinant DNA
4. insert the recombinant DNA into a bacterium
5. culture and grow the bacteria
Restriction Enzymes - answer✔✔a type of endonuclease that cuts up DNA
*normally leave "sticky ends" of DNA
Restrictions sites (recognition sequences) - answer✔✔short nucleotide sequences that are
recognized by restriction enzymes as the place to cut DNA
normally palindromes
sticky ends - answer✔✔cut DNA that is left with short, single stranded sequences on both sides
of the fragments
DNA ligase - answer✔✔seals the two strands of "sticky ended" DNA together by catalyzing
phosphodiester bonds between the single-stranded breaks.
**results in recombinant DNA
Electroporation - answer✔✔makes a cell competent to pick up DNA from the environment by
applying an electrical shock to it, which increases membrane permeability.
RecA proteins - answer✔✔competent cells are engineered to lack these proteins, which may try
to otherwise destroy the inserted sequence or attempt to correct the recombinant DNA
Reverse Transcriptase - answer✔✔turns single-stranded RNA into double-stranded DNA
it is an RNA-dependednt DNA polymerase
Complementary DNA (cDNA) - answer✔✔the DNA produced by reverse transcriptase and the
RNA template
constructing cDNA - answer✔✔1. apply s short poly-T primer to single-stranded RNA
2. add reverse transcriptase and 4 nucleotides
3. add RNaseH enzyme to cut up the RNA and regenerate the RNA primers
4. add DNA polymerase and DNA ligase to synthesize a new strand
cloning vector - answer✔✔provides a means for transferring a gene of interest to a host
organism
"good" cloning vectors include - answer✔✔an origin of replication
a selectable marker
unique restriction sites (polylinkers or multicloning sites)
Plasmid - answer✔✔*most common cloning vector*
a self-replicating piece of extrachromosomal DNA found in prokaryotes
-mid-sized range (20,000 base pairs or less)
pBR322 and pUC19
bacteriophage - answer✔✔virus that infects bacteria
carry smaller fragments between 9,000-25,00 base pairs
cosmid - answer✔✔a hybrid between a plasmid and a phage
carry large fragments between 30,00 and 47,000
artificial chromosomes - answer✔✔synthetic chromosomes that contain fragments of DNA
integrated into a host chromosome
1. P1 artificial chromosomes (PACs)
2. Bacterial artificial chromosomes (BACs)
3. yeast artificial chromosomes (YACs)
**carry up to 1 MILLION bp
DNA analysis - answer✔✔used to determine the location of genes, differences in genes, and
where and when genes are expressed
Includes...
1. PCR
2. gel electrophorese
3. restriction fragment analysis
4. blotting techniques
Polymerase chain reaction (PCR) - answer✔✔quickly and easily makes copies of even a very
small amount of DNA.
targets the DNA with the following:
1. Primers
2. thermostable DNA polymerase (taq polymerase)
3.deoxyribonucleotide triphostates (dATP, dCTP, dGTP, dTTP)
**the solution is heated to 90 to 50 to 70 and then again
Gel electrophoresis - answer✔✔a technique that separates nucleic acids and proteins on the basis
of their size and electrical charge, using a gel that is a polymer
then stained with ethidium bromide for DNA to be visible
Restriction fragment length polymorphism (RELP) - answer✔✔a tool for genetic fingerprinting,
genome mapping, localization of the genes, paternity testing, and microbial classification.
STR analysis - answer✔✔compares genomes based on the number of short tandem repeats
Ribotyping - answer✔✔variation of RELP that compared ribosomal RNA
Blotting - answer✔✔technique that combines electrophoresis and hybridization to detect specific
DNA fragments
Southern Blotting - answer✔✔uses radioactive DNA hybridization probes and autoradiography
to identify the presence or absence of a specific gene
Autoradiography - answer✔✔method for detecting radioactively labeled molecules
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