Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
15 Cards in this Set
- Front
- Back
How can we study the function of a gene?
|
1. gene cloning: which allows the study of the gene in separation from the remainder of the genome.
2. gene inactivation: allows the study of the cellular/organismal effect of loss of gene function. |
|
How do you isolate and propagate a piece of DNA (for example a gene)?
|
With a restriction enzyme that cleaves DNA at a specific sequence (usually palindromic)
|
|
Why do bacteria contain restriction-modification systems?
|
Many bacteria contains restriction-modification systems to "restrict" invasion by foreign DNA
|
|
What are the three elements that plasmid vectors contain?
|
1. a cloning site where the foreign DNA fragment can be inserted
2. a drug-resistance gene (which destroys antibiotics)-in this case ampicillin- to allow selective growth of the host cell 3. A replication origin to allow the plasmid to replicate in the host cell. |
|
what is the first thing we do before we insert foreign DNA?
|
we must first a restriction enzyme to cleave the vector at the cloning site. Foreign DNA containing the sequence we wish to clone is digested with EcoRI and then mixed with the cleaved vector.
|
|
What happens after the restriction enzyme cleaves to the vector at the cloning site?
|
The sticky ends of the foreign and plasmid DNA molecules hybridize and then are sealed into phosphodiester linkages by the enzyme DNA ligase, creating a recombinant plasmid. Each of these recombinant plasmids contains the inserted DNA fragment, an ampicillin resistance gene, and a replication origin.
|
|
What is the importance of the replication origin for plasmid cloning?
|
The replication origin allows the plasmid to replicate by using the host cell's enzymes.
|
|
How do you know your bacterial clone contains your plasmid?
|
You analyze by restriction digestion
|
|
How do you obtain a specific insert DNA (for example a gene)?
|
1. From a DNA source: PCR, Genomic library
2. From an RNA source: RT-PCR to create a cDNA, cDNA library |
|
How do you create a copy DNA (cDNA) clone from mRNA?
|
First you have a mRNA and a primer (oligo-dT) below the 3' that will anneal to all mRNAs. Then with reverse transcriptase and dNTPS the cDNA is formed. RNase H is used which degrades RNA in RNA: (DNA hybrid). DNA oligo and DNA ligase is added at the 3' end and DNA poly and primer will anneal to DNA oligo forming the ds-cDNA.
1. ligate into vector 2. transform to bacteria 3. select |
|
How do you create a genomic library?
|
1. Chop up genomic DNA with restriction enzyme
2. Ligate with plasmid 3. make multiple plasmids, each with different DNA inserts 4. Transform to bacteria, select - Each clone will have a plasmid with a specific genomic insert - Different clones contain different inserts |
|
How do you find the colony that contains a plasmid with YOUR gene of interest in a library?
|
By colony hybridization where 32P- labeled probe (DNA or RNA) hybridizing to your gene of interest. Then you wash away labeled DNA that does not hybridize to DNA bound to filter and perform autoradiography.
|
|
What can you do with your gene clone?
|
1. Express protein (for example in bacteria): For studies of protein function/structure.
2. Express gene, or mutant gene, in organism of origin: For studies of gene expression and/or function. and Gene therapy. |
|
How can we study the function of a gene?
|
1. Gene cloning: allows study of the gene in separation from the remainder of the genome
2. Gene inactivation: Allows study of the cellular/organismal effect of loss of gene function |
|
what is the process for gene inactivation in mice?
|
1. Create knock-out mouse embryonic stem (ES) cells by homologous recombination/drug selection
2. Inject recombinant ES cells into blastocyst and transfer to pseudo-pregnant mouse 3. Back cross with wt mouse, screen for heterozygotic mice 4. Mate heterozygotic mice 5. Screen progeny for homozygotic mice |