Term
| DNA cloning and recombinant |
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Definition
donor DNA cut up by restriction endonucleases -> recombinant DNA molecules are formed -> transferred to bacteria (put in plasmids) -> divides into clones!
epigenetics doesn't exist in bacteria because bacteria don't make histones - naked DNA |
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Definition
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Term
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Definition
| special enzyme that makes cDNA from RNA; originally isolated from retrovirus |
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Term
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Definition
DNA copies of mRNA products (made by reverse transcriptase) - more useful for isolation of protein-coding genes (exons) - (better predictor of poly-peptide sequence)
-cDNA used because RNA is less stable than DNA |
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Term
| polymerase chain reaction (PCR) |
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Definition
simply used to exponentially amplify given DNA sequences
-isolates specific DNA of interest by finding the DNA region by complementary binding of specific short primers to the ends of that sequence which guide the replication process
(powerful technique to isolate specific genes or DNA fragments when there is prior knowledge of the sequence to be amplified)
-can tell exact structure of an exon |
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Term
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Definition
used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase → used to detect RNA expression levels
-useful to look in tissues - see if the gene is in other tissues or mutations in other organisms |
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Term
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Definition
| the use of recombinant DNA techniques to alter an organism's genotype and phenotype |
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Term
| genetic engineering in yeast |
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Definition
| easy because they found they can put something in the plasmid and it will combine into the genome (only eukaryote that does this) -> just put DNA of interest into yeast plasmid and it integrates in yeast chromosomes by homologous recombination with the resident gene by single or double crossover |
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Term
| genetic engineering in plants |
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Definition
Ti plasmid: natural plasmid from soil bacteria wants to grow in plants -> causes crown gall disease which infects the plant and produces uncontrolled growths
-replace T-DNA in Ti Plasmid with genes of interest and marker -> inserts gene of interest into plant (strip away epigenetics) |
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Term
| genetic engineering in mice |
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Definition
1. Constructing recombinant DNA for gene targeting into embryonic stem (ES) cells (knockout gene)
2. The 3 possible ways the recombinant DNA will insert into the genome of the ES cells: targeted insertion, random insertion, no insertion
3. Selecting the ES cell with the correct (targeted) insertion
4. Injecting the targeted ES cells into embryos to generate chimeric mice (put knockout gene in blostocyte (embryo) -> find chimeric mouse in F1 generation)
5. Mating the chimeric mouse and the generation of a “germ line” homozygous mouse (cross chimeric mouse with recessive -> 1/2 brown (1/2 of this 1/2 will be homozygous for this mutation)
-PCR used to determine who carries the mutation |
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Term
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Definition
| gene is inactivated by substituting an inactive gene for the normal gene |
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Definition
| the substitution of an engineered gene for the gene originally in the yeast cell |
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Term
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Definition
remove cells from the a patient, adding a gene to these cells, and place the cells back into the patient
• Has worked sufficiently in SCIDs (sever combined immunodeficiency) → no T-cells, no immune defense
• Take white blood cells and put in the correct gene (retrovirus that is altered so it can not reproduce, designed to integrate into the genome that bring the gene for the missing enzyme)
• Put into the body of the patient, now the person has WBC’s that have a normal immune response
• Every 4-8months they have to go through this procedure again
Cystic fibrosis: breath in virus to infect lung cells so they can make chlorine channels → still has to be done routinely |
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Term
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Definition
| have restriction sites - added to the ends of the cDNA or products of PCR to make cloning efficient |
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Term
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Definition
cDNA library: simpler, contains message sequences only (start with mRNA); screen with nucleic acid or cross-species hybridization; matters what tissue it comes from
Genomic library: more complex, your entire genome (start with DNA); can screen with cDNA
-compare both libraries to get location of introns/exons |
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Term
| dideoxynucleotides (ddNTPs) |
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Definition
| has ability to block continued DNA synthesis -> lacks 3' & 2' -hydroxyl group -> blocking DNA synthesis at the point of addition (contain fluorescent tags) - each synthesized DNA strand is terminated at a specific base by a ddNTP with a specific color |
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Term
| dideoxy sequencing method |
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Definition
| a cloned DNA segment can be sequenced by characterizing the end bases of a serial set of truncated synthetic DNA fragments, each terminated at different positions corresponding to the incorporation of a dideoxynucleotide |
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Term
| DNA denaturation and hybridization |
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Definition
DNA hybridization: single-strand pairing
DNA denaturation: necessary before PCR or probing |
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Term
| cross-species hybridization |
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Definition
| if someone has cloned a specific protein in one species you can use this clone to find the protein in another species |
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Term
| screening a library with a hybridization probe |
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Definition
-all DNA molecules must be single stranded by heating
-single stranded probe labeled radioactively or chemically sent out to find complimentary target sequence
-DNA is denatured and then bathed in solution of single-stranded probe which allows it to stick to the membrane |
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Term
| screening an expression library with an antibody |
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Definition
antibody is made by injecting a mammal with the protein of interest, the mammal's immune system will create an antibody for that specific protein (find in blood of animal) -> put antibody on plate of cDNA's -> will bind to specific protein product of the gene of interest
-handy if you don't know the gene of interest but know the protein of interest!
-to detect location of 1st antibody -> 2nd antibody binds to the 1st |
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Term
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Definition
insert cDNA into a protein coding region on the bacteria plasmid so that the bacteria expresses the genes that are turned on
combination of clones cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcription of the organism
used when a specific gene is active in specific type of tissue |
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Term
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Definition
RNA analysis
-used to determine whether a specific gene is transcribed in a certain tissue or under certain environmental conditions or to determine size of the mRNA
-don't need to denature mRNA
-probe with cDNA
-should see the band from every tissue that expresses that gene (if different lengths - alternative splicing!) |
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Term
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Definition
DNA analysis
o When genomic DNA is blotted against a cDNA probe → every fragment that contains an exon sequence will hybridize to the cDNA (end up with lots and lots of bands) |
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Definition
| comparing sick people to healthy people (want to determine if people have the disease) |
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