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15 Cards in this Set

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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