FOB EXAM 4

Why is cDNA used to clone eukaryotic genes into prokaryotic cells?

it has no introns

What is the function of reverse transcriptase?

to turn RNA into DNA

What template does reverse transcriptase use to synthesize cDNA?

mRNA

In the formation of cDNA when mRNA is transcribed to create cDNA what activity is this?

RNA dependent DNA polymerase

In the formation of cDNA when the mRNA is degraded what type of activity is this?

RNase

In the formation of cDNA when a second cDNA strand is synthesized what activity is this?

DNA dependent DNA polymerase

What are the steps of cloning?

1. Isolate the gene and clone it into a vector

2. Cut DNA and plasmid using the same RE

3. Join fragments using DNA ligase

4. Transformation (bacteria takes plasmid)

5. identify the bacteria that has the plasmid

6. screen for bacteria containing the gene of interest

What does PCR do?

amplify DNA without cloning it

What are the steps of PCR?

1. Unwinding by heating

2. add primers in excess

3. Taq polymerase

In PCR why are the primers added in excess?

to make sure the parents won’t come back

IN PCR what is added along with Taq?

DTPs

Where do restriction enzymes cleave the DNA double helix?

They cut the nucleic acid backbone from within

What do restriction enzymes recognize in DNA?

palindromic sequence

What is the advantage of using directional cloning?

DNA inserts in one orientation

What can happen if a vector is cleaved with the same restriction enzyme and ligated without an insert?

The vector religates

What does each individual spot on a DNA microarray represent?

a known DNA sequence or gene

What needs to be in frame in order to clone/express a gene?

the start codon and gene of interest

If you would like to investigate the effect of knocking out the expression of a receptor by transfection with a microRNA specific to the receptor’s mRNA, it is best to target the microRNA to the….

cytosol

What is the purpose of an expression vector?

produce protein from foreign DNA

What is the correct order of the components in the expression vector?

1. promoter

2. ATG start codon

3. (His)6 tag

4. EK cleavage sequence

5. Multiple cloning site

What is the function of the promoter?

binding/start signal for RNA polymerase

Why is the ATG start codon necessary?

signals the beginning of translation

What is the purpose of the (His)6 tag?

allows the protein to bind to a nickel affinity column so it can be purified

What is the function of the enterokinase (EK) cleavage sequence?

allows (His)6 tag to be cleaved off

What is the purpose of the multiple cloning site (MCS)?

contains multiple restriction enzyme recognition sites where the gene can be inserted

An expression vector must have a ________ binding site and start codon.

ribosomal

In EMSA if you have a band migrating higher than the bare DNA that means there is a

DNA-protein complex

What is a stable transfection?

DNA integrated into a chromosome

What is a transient transfection?

DNA delivered into the nucleus but not integrated

What vector integrates into the host and infects dividing cells?

retroviral

What vector doesn’t integrate into the host and produces temporary gene expression?

adenoviral

What vector integrates into the vector and infects dividing and nondividing cells?

Lentiviral

What is insulin?

A chain + B chain covalently held together by inter disulfide bond