Flashcards in Genetic Engineering Deck (27):
Human manipulation of an organism's DNA
Current Methods of Genetic Engineering include recombinant DNA technology (rDNA):
transplanting a gene (trans gene) from one organism to another
-ex. insulin production
-created using molecular cloning
DNA of interest is inserted into a cloning vector.
What would you use the final clone for? What kind of experiments would you use it in?
-Use the gene product
-Figure out what gene product does:
*test the biochemistry
*use a model organism
*localize protein with a tag
-Diagnoses of disease, genotype
-Make a mutation
-Sequence gene of interest
-a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.
-contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together.
General Cloning Strategy
1. Isolate DNA of interest from an organism
2. Cut the DNA with a restriction enzyme
3. Cut the cloning vector with the same restriction enzyme
4. Ligate piece into a cloning vector to make a recombinant DNA molecule
5. Transform the recombinant DNA into a host (like E. coli), the host cell will make identical copies called clones.
Products of restriction enzymes:
Relationship between restriction enzymes and ligase:
Opposing roles; Restriction enzymes cut and Ligase seals
A collection of plasmids (clones) in which each plasmid theoretically contains a different piece of DNA.
General Steps to make a Genomic Library
1. DNA gets extracted from an organism of interest
2. DNA is then cut up into small pieces using restriction enzymes
3. Each piece gets ligated into cloning vectors
4. Library is formed
Importance of Genomic Libraries
-Can locate a specific sequence
-Assists with diagnostics
-Sequences by "shot-gunning":
*Screens, ie. suppressor screening by transforming a library into a mutant and checking for revertants to the wild type phenotype.
Goal: to find a DNA sequence in a pool of DNA
-Use a DNA probe and Caveat
Screening a Genomic Library
a labeled segment of DNA or RNA used to find a specific sequence of nucleotides in a DNA molecule.
-may be synthesized in the laboratory, with a sequence complementary to the target DNA sequence.
General Steps in making a cDNA Library
1. RNA is extracted from an organism of interest
2. RNA is reverse transcribed into cDNA
3. Each cDNA is cloned into cloning vectors
4. Each library clone theoretically has a different cDNA
Importance of cDNA Librares
-Detects sequence of interest
-Allows you to tell the difference between exons and introns
-Can look for splice variance (Alternative Splicing)
It is DNA synthesized from a mRNA template in a reaction catalysed by the enzymes reverse transcriptase.
-often used to clone eukaryotic genes in prokaryotes.
Amplify a DNA segment of interest.
-example: amplify a portion of DNA to clone into a cloning vector.
PCR--polymerase chain reaction
Uses for PCR
-Sequence an entire genome/transcriptome
-quantify a transcript
-Make mutations in a gene of interest
*M vs. F
*++ vs. Mutant
*Diseased vs. Healthy
Measures the increase in the amount of PCR product during the thermal cycling/rxn.
-Measure a specific DNA as it's amplified.
-Therefore, can measure abundance of transcript in a sample.
Real-time PCR aka Quantitative PCR (qPCR)
Reasons for mutagenesis:
-Suppresor mutations ("fix it")
-Study the mutation: see if it's advantageous, identify a disease-causing mutation, identify the "important" amino acid in a polypeptide to understand the role of a particular amino acid in a polypeptide.
2 Major types of mutagenesis; ex) they add Mn+2 instead of Mg+2, which causes Polymerase to have reduced fidelity (will add mistakes)
1. Site Directed Mutagenesis
2. Random Mutagenesis
Intentionally mutate 1 specific nucelotide.
-include primer with single nucleotide change.
Site Directed Mutagenesis
Create random mutations in DNA of interest
Overall goal: To detect a specific DNA sequence in a sample of DNA
*compare homologous genes
*analyze intron organization
*diagnostics of diseases
*look for methylated sequences
Molecular Analysis of DNA by Southern Blotting
General Steps of Southern Blotting:
1. Isolate DNA from an organism of interest.
2. Cut DNA using fragments using restriction enzymes
3. Separate DNA fragments with gel electrophoresis
4. Transfer DNA from gel to a membrane ("blot")
*DNA will end up in the same position as it appeared on gel.
5. Incubate blot with labeled probe complementary to DNA of interest.
6. Visualize probe-band with DNA of interest
Example of Southern Blotting: RFLP diagnosis of Sickle Cell Anemia