krit.club logo

Biotechnology: Principles and Processes - Restriction Enzymes and Vectors

Grade 12CBSEBiology

Review the key concepts, formulae, and examples before starting your quiz.

🔑Concepts

•

Restriction Endonucleases: These are 'molecular scissors' that cut DNA at specific locations called recognition sequences. The first restriction endonuclease discovered was HindIIHindII.

•

Palindromic Nucleotide Sequences: Restriction enzymes recognize specific palindromic sequences in DNA where the reading frame is the same on both strands in the 5′→3′5' \rightarrow 3' direction, such as 5′−GAATTC−3′5' - GAATTC - 3' and 3′−CTTAAG−5′3' - CTTAAG - 5' for EcoRIEcoRI.

•

Sticky Ends vs. Blunt Ends: Enzymes like EcoRIEcoRI leave overhanging single-stranded stretches called 'sticky ends', which facilitate the action of DNA ligaseDNA\ ligase by forming HH-bonds with complementary cut DNA.

•

Cloning Vectors: DNA molecules used as vehicles to carry foreign DNA into a host cell. Plasmids (e.g., pBR322pBR322) and bacteriophages are commonly used due to their high copy number.

•

Origin of Replication (oriori): A specific DNA sequence responsible for initiating replication and controlling the copy number of the linked DNA.

•

Selectable Markers: Genes that help in identifying and eliminating non-transformants and selectively permitting the growth of transformants (e.g., genes encoding resistance to antibiotics like ampRamp^R or tetRtet^R).

•

Insertional Inactivation: A technique where a recombinant DNA is inserted within the coding sequence of an enzyme (like β\beta-galactosidase) or an antibiotic resistance gene, causing its inactivation to help distinguish recombinants.

•

Gel Electrophoresis: A technique to separate DNA fragments based on size. DNA is negatively charged and moves towards the AnodeAnode (+). Fragments are visualized using Ethidium bromideEthidium\ bromide (EtBrEtBr) under UVUV light.

📐Formulae

Frequency of a specific restriction site=(14)n\text{Frequency of a specific restriction site} = \left(\frac{1}{4}\right)^n where nn is the number of base pairs in the recognition sequence.

Velocity of DNA fragment (v)∝1log(Molecular Weight)\text{Velocity of DNA fragment (v)} \propto \frac{1}{\text{log}(\text{Molecular Weight})}

EcoRI Nomenclature=E (Escherichia)+co (coli)+R (RY 13)+I (First discovered)\text{EcoRI Nomenclature} = \text{E (Escherichia)} + \text{co (coli)} + \text{R (RY 13)} + \text{I (First discovered)}

💡Examples

Problem 1:

A foreign DNA is ligated at the BamHIBamHI site of the vector pBR322pBR322. What will be the phenotype of the resulting transformants regarding antibiotic resistance?

Solution:

The transformants will be resistant to ampicillinampicillin but sensitive to tetracyclinetetracycline.

Explanation:

In pBR322pBR322, the BamHIBamHI recognition site is located within the tetRtet^R (tetracycline resistance) gene. Inserting foreign DNA at this site causes 'insertional inactivation' of the tetRtet^R gene, while the ampRamp^R gene remains functional.

Problem 2:

Identify the palindrome sequence for the enzyme EcoRIEcoRI and show where the cleavage occurs.

Solution:

5′−G↓AATTC−3′5' - G \downarrow AATTC - 3' and 3′−CTTAA↑G−5′3' - CTTAA \uparrow G - 5'

Explanation:

Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite strands. For EcoRIEcoRI, it cuts between GG and AA on both strands, creating 'sticky ends' (AATTAATT).

Problem 3:

Why is the oriori sequence considered the most important part of a cloning vector?

Solution:

It controls the copy numbercopy\ number and initiates replication.

Explanation:

The oriori (Origin of Replication) is the sequence from where replication starts. If one wants to recover many copies of the target DNA, it should be cloned in a vector whose oriori supports a high copy number.

Restriction Enzymes and Vectors - Revision Notes & Key Diagrams | CBSE Class 12 Biology