Term
| what is recombinant DNA technology? |
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Definition
| construction of DNA from 2 species |
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Term
| what is genetic engineering? |
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Definition
| applied manipulation of genes of DNA towards particular problem |
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Term
| what 2 basic principles of DNA are exploited for genetic technology? |
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Definition
1. single stranded DNA will hybridize with complementary strands
2. Certain binding proteins bind to specific nucleotide sequences |
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Term
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Definition
DNA sequene inserted into small genome (such as plasmid).
allowed to multiply in/with host cell (aka gene cloning) |
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Term
| what are the three main sources of DNA for cloning? |
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Definition
1. genomic DNA (from normal DNA, includes introns)
2. artificially synthesized DNA (up to 100 nucleotides)}
3. Complementary DNA (from mRNA) |
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Term
| what is used to make complementary DNA? what is the source? |
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Definition
viral reverse transcriptase
from retroviruses |
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Term
| what are the enzymes that cut DNA at specific sequences? |
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Definition
| restriction endonucleases |
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Term
| what type of sequence often makes up a 'sticky end'? |
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Definition
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Term
| what is the source and original role of restriction endonucleases? |
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Definition
| in bacteria to combat phage. cuts phage dna |
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Term
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Definition
| the amplifying and purifying of specific genes |
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Term
| what is a restrcition site? |
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Definition
| the site on DNA where a specific restriction enzyme cuts the DNA. |
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Term
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Definition
| the looses bonding of 2 cleaved pieces at complementary sticky ends |
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Term
| what enzyme used to strengthen the bond between hybridized DNA? |
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Definition
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Term
| what enzyme used in PCR? why? |
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Definition
Taq polymerase
polymerase from extremophile bacteria, able to withstand high heat |
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Term
| what is a cloning vector? examples. |
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Definition
a small genome into which a foregin fragment of DNA is inserted.
examples: bacteria plasmid, yeast plasmid, phage DNA |
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Term
| what are properties desirable in an ideal cloning vector? |
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Definition
1. small in size
2. makes many copies of itself in host cell
3. carries known restriction sites
4. provides for selectability (ex. antibiotic resistance) |
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Term
| what are 3 ways that recombinant DNA can enter host cells? |
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Definition
1. transformation
2. transduction (via phage holding bacterial DNA)<3> phage infection (using phage DNA) |
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Term
| what is an expression vector? |
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Definition
a vector in which the inserted gene is expressed (protien made).
often recombinant DNA is inserted in gene with known promoters that can be turned on/off |
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Term
| what is a genetic library? |
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Definition
| a set of clones, each w/a different section of the donar's genome |
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Term
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Definition
| a short strand of DNA or RNA complementary to the section of interest that is made of radioactive nucleotides |
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Term
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Definition
1. homologous genes of related organisms
2. reverese transcription of a protein
3. cDNA |
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Term
| what are the problems with reverese transcribing a protein to obtain a DNA probe? |
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Definition
1. only exons will be coded
2. b/c of multiple codons for some a.a will be diff. possible reverese translations |
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Term
| what is an expression vecto library? |
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Definition
| a library of clones that are actively making proteins |
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Term
| what would be used to screen expression vector clones for a certain protein product? |
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Definition
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Term
| what are antibodies in regards to expression vector cloning? |
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Definition
| protein molecules that bind specifically with other protein molecules |
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Term
| what is the difference between a southern and northern blot? |
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Definition
southern - DNA
northern - RNA |
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Term
| what is the general procedure of a southern blot? |
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Definition
| DNA fragments placed in wells in gel besided known fragments. electric charge applied, fragments travel. transferred to papaer and stained or probed for certain DNA sequences |
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Term
| what is a molecular marker? what is a common example? |
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Definition
an identifyable sequence on the DNA
such as a restriction site |
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Term
| what is restriction mapping? |
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Definition
| the mapping of restriction sites on a csome |
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Term
| what are dideoxy nucleotides and how are they used to sequence DNA? |
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Definition
nucleotides with no oxygen on 3' carbon.
are unable to form phosphodiester bonds so end fragment being synthesized, result in fragments of specific lengths |
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Term
| how many open reading frames are possible in one double strand of DNA? |
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Definition
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Term
| what plasmid is often used in the engineering of genetically modified organisms? where is the plasmid derived? |
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Definition
| T1 plasmid from Agrobacterium tumefaciens |
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Term
| how are transgenic animals created? |
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Definition
fertilized embryos injected w/ many copies of the gene
-some integrated by embryo and can be passed on as Mendellian allele |
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Term
| what is 'Germ-line' therapy and somatic cell theory? |
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Definition
Germ-line - transgene passed on
somatic - in somatic cells, not passed on |
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Term
| what disease was somatic cell therapy successfully performed on (in some cases)? |
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Definition
| Severe Combined Immune Disorder (SCID) - 'bubble boy' disease |
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Term
| what is the resolution of cytogenic or recombination based mapping? |
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Definition
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Term
| what are RFLP's? how are they used? |
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Definition
Restriction Fragment Length Polymorphisms
- used to map restriction sites on idividuals for higher resolution mapping |
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Term
| how can RFLPs be used to find location of allele on csome? |
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Definition
obtain RFLPs for individuals in pedigree
- see which restriction sites are associated with allele |
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Term
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Definition
| simple sequence length polymorphism - small sequences repeated in tandem |
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Term
| where are SSLPs often found? |
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Definition
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Term
| what are 2 types of SSLP? what is the difference? |
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Definition
minisatellite markers - ~33 bp repeats
microsatellite markers - 2 bp repeats |
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Term
| what are VNTR's? how used? |
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Definition
| SSLP's that are Variable Number of Times Repeated - differ from individual to individual, often called DNA fingerprints |
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Term
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Definition
| Randomly Amplified Polymorphic DNA - short PCR probe used to amplify a random section of DNA |
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Term
| what is In-Situ hybridization mapping? |
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Definition
| fluorescentally labelled DNA probe from cloned fragment is allowed to bind w/intact csome to see where section is |
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Term
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Definition
| Fluorescent In-Situ Hybridization |
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Term
| what is the resolution of FISH? |
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Definition
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Term
| how is a physical map obtained? |
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Definition
| clones sequenced individually and placed in order |
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Term
| what are 2 general types of repetitive fragments in eukaryote genomes? |
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Definition
Tandem Repeat Arrays
Mobile Genetic Elements |
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Term
| what are some examples of Tandem Repeat Arrays in eukaryotes? |
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Definition
| minisatellites (VNTRs), microsatellites, telomeres, repeated genes (histone genes) |
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Term
| what is a mobile genetic element? |
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Definition
| gene sequence that propagates itself within the genome. copies itself and moves around to new locations (transposition) |
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Term
| how much of human genome is made of mobile genetic elements? what is the likely origin? |
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Definition
40%
likely from retroviruses |
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Term
| what is aeuploid change in csome #? aneuploid? |
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Definition
euploid - has complete sets
aneuploid - incomplete sets |
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Term
| why does odd ploidy lead to sterility? |
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Definition
|
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Term
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Definition
| spontaneous doubling of genome (polyploid mutation) seen in plants, resulting in 'instant speciation' |
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Term
| how many csomes does a human with a monosomy mutation have? |
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Definition
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Term
| how does aneuploidy arise? |
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Definition
| non-disjunction of meiosis I or II |
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Term
| what are the only monomies that survive in humans? |
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Definition
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Term
| what trisomies possible in humans? |
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Definition
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Term
| what are the 4 types of csomal mutation? |
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Definition
Deletion
Duplication
Inversion
Translocation |
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Term
| what type of csomal mutation is usually homozygous lethal? |
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Definition
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Term
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Definition
| the expression of a recessive allele over a deletion |
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Term
| how may duplications have played a role in evolution of new genes and gene families. |
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Definition
| having many copies of a gene allows some copies to mutate without extreme consequene on organism |
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Term
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Definition
| when related species show similarities in csome structure, revealing common ancestory |
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Term
| what proporion on pregnancies spontaneously abort b/c of csome mutations? |
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Definition
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