Recombinant DNA Technology & Library Construction

Question 1

What is recombinant DNA technology?

Answer 1

It is a set of techniques used for recombining genes from various sources in vitro which are transferred to a host cell where it may be expressed.

Question 2

Requirements for recombinant DNA technology (6)

Answer 2

1. gene/gene source
2. vector
3. enzymes (restriction endonuclease, ligase)
4. transformation method
5. host cell
6. selection method

Question 3

General Steps of recombinant DNA technology

Answer 3

1. Restriction enzymes cut both the vector and the donor DNA at specific sites, creating overhangs
2. the donor DNA is then inserted into the vector through a transformation method, producing recombinant vectors
3. the recombinant vectors are inserted into a host cell to allow replication
4. a method for selection is determined to detect successful transformation

Question 4

enzymes isolated chiefly from prokaryotes that recognize specific sequences within the double-stranded DNA

Answer 4

restriction endonucleases

Question 5

mechanism of RE

Answer 5

cuts at specific restriction site (short nucleotide sequence)

Question 6

How do RE protect the bacteria?

Answer 6

It prevents foreign DNA from infecting/invading by degrading it

Question 7

How do bacterial cells escape restriction digestion by their own REs?

Answer 7

the host DNA is modified by methylation (via methylase) which prevents recognition of the RE

Question 8

Used RE type for recombinant DNA technology

Answer 8

type II

Question 9

RE Type I

Answer 9

moves along the DNA before cutting the strand, and releasing a number of nucleotides where the cut is made

Question 10

RE Type II

Answer 10

recognizes a specific target sequence and break the DNA within or close to the recognition site

Question 11

RE Type III

Answer 11

intermediate of RE Types I and II

Question 12

RE Type IV

Answer 12

targets methylated DNA

Question 13

two possible resulting restriction fragments

Answer 13

1. blunt ends
2. overhangs

Question 14

joining linear DNA

Answer 14

ligation

Question 15

ligation enzyme

Answer 15

DNA ligase

Question 16

a DNA molecule that carries the foreign DNA into a host cell, replicates inside and produces copies of itself and the foreign DNA

Answer 16

cloning vector

Question 17

3 features of a cloning vector

Answer 17

1. sequence for replication
2. cloning site where the foreign DNA is inserted
3. method for selection for the cells containing the vector with the foreign DNA

Question 18

difference between a cloning vector and an expression vector

Answer 18

they’re the same, except the expression vector has the initiator and terminator genes for transcription to allow for gene expression of the cloned gene

Question 19

uptake of DNA by bacterial cells

Answer 19

transformation

Question 20

give at least 3 transformation methods

Answer 20

1. chemical induction
2. chemical induction + heat shock
3. electroporation
4. gene gun
5. microinjection
   etc.

Question 21

Mechanism of the antibiotic resistance screening

Answer 21

• gene is inserted in the cloning site located within the antibiotic resistance gene
• plating in the antibiotic, empty vectors should survive whereas cells containing the recombinant vectors should not survive

Question 22

mechanism of the blue-white screening

Answer 22

• the foreign gene is inserted in the cloning site within the lacZ gene
• if the transformation is unsuccessful, the lacZ gene remains active, producing the ß-galactosidase where in the presence of gal-X produces blue colonies
• recombinant colonies contain the inactive lacZ gene, thereby restricting production of the ß-galactosidase, and in the presence of gal-X produces white colonies

Question 23

collection of DNA fragments derived from the genome of an organism and randomly cloned into suitable cloning vectors

Answer 23

DNA Library

Question 24

differentiate genomic library vs cDNA library

Answer 24

Genomic Library contains fragments of all the DNA sequences present in a cell or organism, whereas the cDNA library contains the entire mRNA sequence of a particular type of cell or tissue.

For the genomic library, DNA is extracted and purified from the sample.
- DNA is digested with RE to produce insertional fragments.
- The fragments are ligated to the vectors (also cut by RE) to produce the recombinant DNA.
- The recombinant vectors are transformed into a host cell and cultured in selective media.
- Then, the selected clones for specific genes can be screened by probing.

For the cDNA library, mRNA is extracted from a cell type followed by reverse transcription to form the cDNA.
- The cDNA is ligated with linkers containing the sites of RE digestion.
- RE cuts at the specific sites, and then can be ligated to the vectors to form the recombinant DNA.
- Just like in the genomic library construction, this is followed by transformation, culture, and screening.

Question 25

Advantages and disadvantages of genomic library.

Answer 25

Genomic library is good for determining the genomic structure and can be isolated from any type of tissue. However, it requires a large number of clones to increase the probability of representing both the coding and the non-coding DNA sequences.

Question 26

advantages and disadvantages of cDNA library

Answer 26

The cDNA library uses short sequences and is therefore easy to sequence. It determines the functional activity of the cells from which the library was constructed, and the clones can be used to directly express the proteins coded by them (if full-length.

However, each library is unique only for the type of cell from which it was sampled, and it does not reflect information about the structure of the gene.

Question 27

Subtractive hybridization of PCR-based libraries

Answer 27

Two PCR-based libraries, one biotin-labeled (driver library) and one unlabeled (tester library), are boiled and allowed to hybridize. Common sequences from both libraries will then be labeled. The biotin-labeled DNA sequences can be removed using avidin, and the remaining sequences would be the DNA sequence unique to the tester library, which can be amplified by PCR.