Recombinant DNA Technology & Library Construction Flashcards

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1
Q

What is recombinant DNA technology?

A

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.

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2
Q

Requirements for recombinant DNA technology (6)

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

General Steps of recombinant DNA technology

A
  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
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4
Q

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

A

restriction endonucleases

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5
Q

mechanism of RE

A

cuts at specific restriction site (short nucleotide sequence)

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6
Q

How do RE protect the bacteria?

A

It prevents foreign DNA from infecting/invading by degrading it

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7
Q

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

A

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

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8
Q

Used RE type for recombinant DNA technology

A

type II

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9
Q

RE Type I

A

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

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10
Q

RE Type II

A

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

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11
Q

RE Type III

A

intermediate of RE Types I and II

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12
Q

RE Type IV

A

targets methylated DNA

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13
Q

two possible resulting restriction fragments

A
  1. blunt ends
  2. overhangs
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14
Q

joining linear DNA

A

ligation

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15
Q

ligation enzyme

A

DNA ligase

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16
Q

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

A

cloning vector

17
Q

3 features of a cloning vector

A
  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
18
Q

difference between a cloning vector and an expression vector

A

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

19
Q

uptake of DNA by bacterial cells

A

transformation

20
Q

give at least 3 transformation methods

A
  1. chemical induction
  2. chemical induction + heat shock
  3. electroporation
  4. gene gun
  5. microinjection
    etc.
21
Q

Mechanism of the antibiotic resistance screening

A
  • 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
22
Q

mechanism of the blue-white screening

A
  • 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
23
Q

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

A

DNA Library

24
Q

differentiate genomic library vs cDNA library

A

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.

25
Q

Advantages and disadvantages of genomic library.

A

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.

26
Q

advantages and disadvantages of cDNA library

A

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.

27
Q

Subtractive hybridization of PCR-based libraries

A

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.