cDNA Synthesis🪱

Question 1

Expression Libraries

Answer 1

each cloned piece of DNA is expressed into a protein by the vector (whether a real gene or not)

a library of dna fragments which was created with an expression vector so that any genes present in the library are expressed.

Question 2

cDNA

Answer 2

a double-stranded DNA copy of mRNA made by reverse transcriptase

Question 3

Do eukaryotes or prokaryotes have more noncoding DNA

Answer 3

Eukaryotes

Question 4

How are expression libraries created for bacteria?

Answer 4

Genomic DNA can be used directly

Question 5

How are expression libraries created for eukaryotes?

Answer 5

DNA must first be made into cDNA

Question 6

How do eukaryotes make cDNA?

Answer 6

1. mRNA is isolated from the organism by binding to a column containing poly(T)
2. Then the mRNA is converted into cDNA using reverse transcriptase
3. an enzyme removes the mRNA part of the mRNA/cDNA heteroduplex and DNA polymerase makes the second strand of DNA

Question 7

What happens after the cDNA is created?

Answer 7

it is ligated into an expression vector

Question 8

How many times can each insert be translated and why?

Answer 8

Each insert can be translated in three different reading frames because the genetic code is a triplet.

Question 9

How many of the three reading frames will produce the correct protein?

Answer 9

ONE

Question 10

Is cDNA cloned in all three reading frames?

Answer 10

Yes, using linkers with several different restriction sites

Question 11

How is the protein of interest identified?

Answer 11

By incubating the library with an antibody to the protein of interest