Tools of Recombinant DNA Technology

BiologyBiotechnology: Principles and ProcessesFor NEET aspirants

The tools of recombinant DNA technology are restriction enzymes, polymerase enzymes, ligases, vectors and a competent host. This page explains how restriction enzymes are named and how they cut palindromic sites to leave sticky ends, how gel electrophoresis separates DNA fragments, what a cloning vector such as pBR322 needs, how recombinants are selected, and how DNA enters host cells, as in NCERT Class 12 Biology. NEET often asks enzyme naming, pBR322 sites and selection of recombinants, so learn each of the tools of recombinant DNA technology exactly.

On this page1Key tools2Restriction enzymes3Palindromes and sticky ends4Gel electrophoresis5Cloning vectors6pBR322 and selection7Plant and animal vectors8Competent host9Exam essentials10Quick revision11Solved examples12Practice

Key Points at a Glance

★ Must learn Key tools: restriction enzymes, polymerase enzymes, ligases, vectors and the host organism.
1963: two enzymes that restrict bacteriophage growth in E. coli were isolated; one adds methyl groups to DNA, the other cuts DNA.
★ Must learn Hind II, the first sequence-specific restriction endonuclease, was isolated five years later; it recognises a specific sequence of six base pairs.
More than 900 restriction enzymes are known, from over 230 bacterial strains.
★ Must learn EcoRI: E = genus Escherichia, co = species coli, R = strain RY 13, I = first enzyme isolated from that strain.
Exonucleases remove nucleotides from DNA ends; endonucleases cut at specific positions within DNA.
★ Must learn Recognition sites are palindromes, such as GAATTC; a staggered cut leaves sticky ends that DNA ligase can join.
Gel electrophoresis: DNA moves to the anode through agarose; smaller fragments move farther; ethidium bromide and UV show orange bands; elution recovers them.
★ Must learn A cloning vector needs an ori, a selectable marker and very few, preferably single, recognition sites for each commonly used restriction enzyme.
pBR322: BamH I and Sal I lie in tet^R; Pst I and Pvu I lie in amp^R; rop codes for proteins of plasmid replication.
★ Must learn Insertional inactivation of $β$ -galactosidase: blue colonies have no insert; colourless colonies are recombinants.
Competent cells: $C a^{2 +}$ treatment, then ice, heat shock at $42^{\circ} C$ , ice. Other routes: micro-injection, biolistics (gene gun), disarmed pathogens.

1. Key Tools of Recombinant DNA Technology

Genetic engineering, or recombinant DNA technology, can be done only with certain key tools.

Tool	Its job
Restriction enzymes	Cut DNA at specific recognition sequences
Polymerase enzymes	Copy DNA (for example, DNA polymerase in PCR)
Ligases	Join the ends of cut DNA molecules
Vectors	Carry the alien DNA into the host and multiply it
Host organism	Takes up the recombinant DNA and multiplies it

Memory Trick Cut, Copy, Paste, Carry, Keep: restriction enzymes cut, polymerases copy, ligases paste, vectors carry and the host keeps and multiplies the DNA.

2. Restriction Enzymes

2.1 Discovery

★ Exam imp In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated.
One of them added methyl groups to DNA; the other cut DNA.
The enzyme that cut DNA was called restriction endonuclease.
The first restriction endonuclease whose action depended on a specific DNA nucleotide sequence, Hind II, was isolated and characterised five years later.
Hind II always cut DNA at a particular point by recognising a specific sequence of six base pairs.
Today more than 900 restriction enzymes are known, isolated from over 230 strains of bacteria; each recognises a different recognition sequence.

Recognition sequence: the specific base sequence that a restriction endonuclease recognises in DNA, and at which it cuts; for Hind II it is six base pairs long.

2.2 Naming restriction enzymes

The first letter of the name comes from the genus.
The second two letters come from the species of the prokaryotic cell from which the enzyme was isolated.
The next letter, if any, comes from the strain.
★ Exam imp Roman numbers after the name show the order in which the enzymes were isolated from that strain of bacteria.

Part of 'EcoRI'	Comes from
E	Genus Escherichia
co	Species coli
R	Strain RY 13
I	First enzyme isolated from this strain

Memory Trick 1 + 2 + strain + order: one letter of the genus, two letters of the species, then the strain, then a Roman numeral for the order of isolation. E + co + R + I = EcoRI from Escherichia coli RY 13.

Extra Depth: Sources of the other enzymes named on this page: Hind II and Hind III, Haemophilus influenzae (strain Rd); BamH I, Bacillus amyloliquefaciens (strain H); Sal I, Streptomyces albus; Pst I, Providencia stuartii; Pvu I and Pvu II, Proteus vulgaris; Cla I, Caryophanon latum.

2.3 Nucleases: exonucleases and endonucleases

Restriction enzymes belong to a larger class of enzymes called nucleases, which are of two kinds.

Exonucleases

Remove nucleotides from the ends of the DNA.

Endonucleases

Make cuts at specific positions within the DNA. Restriction endonucleases belong here.

Memory Trick Exo = exit, Endo = inside: exonucleases nibble at the exits (ends); endonucleases cut inside the molecule.

2.4 How a restriction endonuclease cuts

It 'inspects' the length of a DNA sequence.
It finds its specific recognition sequence.
It binds to the DNA at that site.
It cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

★ Exam imp Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

2.5 Palindromes in DNA

A palindrome in words is a group of letters that reads the same forward and backward, for example, MALAYALAM.
A word palindrome reads the same in both directions along one line.

★ Very important Palindrome in DNA: a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same.

5^{'} - GAATTC - 3^{'}

3^{'} - CTTAAG - 5^{'}

Read in the $5^{'} \to 3^{'}$ direction, both strands give GAATTC.
This is also true when both are read in the $3^{'} \to 5^{'}$ direction.

Tips and Tricks Check a palindrome in two moves: read the top strand from left to right (

5^{'} \to 3^{'}

), then read the bottom strand from right to left (also

5^{'} \to 3^{'}

). If both readings match, the site is palindromic. GAATTC passes; GAATTG fails, because its partner strand read

5^{'} \to 3^{'}

is CAATTC.

2.6 Sticky ends

Restriction enzymes cut the strand a little away from the centre of the palindrome site, but between the same two bases on the opposite strands.
This leaves single-stranded portions at the ends: overhanging stretches on each strand.
EcoRI cuts between G and A only where GAATTC is present, so each end carries the single-stranded overhang AATT.

★ Very important Sticky ends: the overhanging single-stranded stretches left by a restriction enzyme. They are 'sticky' because they form hydrogen bonds with their complementary cut counterparts; this stickiness helps the enzyme DNA ligase act.

Test yourself: hide labels

Figure 1: Steps in the formation of recombinant DNA by the restriction endonuclease EcoRI. The staggered cut between G and A leaves matching sticky ends on vector and foreign DNA, which then join.

Memory Trick G, cut, AATTC: EcoRI cuts after G in GAATTC on both strands, so every EcoRI end has the overhang AATT.

2.7 Joining DNA from different sources

Restriction endonucleases are used in genetic engineering to form recombinant DNA molecules: DNA composed of DNA from different sources or genomes.
★ Exam imp When cut by the same restriction enzyme, the DNA fragments have the same kind of sticky ends, and DNA ligases can join them end to end.
Unless the vector and the source DNA are cut with the same restriction enzyme, the recombinant vector molecule normally cannot be created.

Test yourself: hide labels

Figure 2: Diagrammatic representation of recombinant DNA technology. One restriction enzyme cuts both foreign DNA and the plasmid, ligase joins them, and transformation puts the recombinant DNA into E. coli, which multiplies it as the cells divide.

The same restriction enzyme cuts both the foreign DNA and the vector DNA (plasmid) at a specific point.
Ligases join the foreign DNA to the plasmid.
A recombinant DNA molecule is formed.
Transformation puts it into the cloning host, E. coli.
The cells divide, and each daughter cell carries the recombinant DNA.

NEET Focus

Statement traps on restriction enzymes: they cut both strands; the cut is a little away from the centre of the palindrome but between the same two bases on both strands; vector and source DNA must be cut with the same enzyme; it is DNA ligase, not the restriction enzyme, that joins the sticky ends.

Quick Recall: tap to check

In which year were the two enzymes restricting bacteriophage growth in E. coli isolated?

1963.

What does the letter R in EcoRI stand for?

The strain, RY 13.

Why are sticky ends called sticky?

They form hydrogen bonds with their complementary cut counterparts.

Key idea

A restriction endonuclease recognises a palindrome, cuts both strands off centre and leaves sticky ends that ligase can join to any DNA cut by the same enzyme.

3. Separation and Isolation of DNA Fragments

Cutting DNA with restriction endonucleases gives DNA fragments.
These fragments are separated by a technique called gel electrophoresis.
★ Exam imp DNA fragments are negatively charged, so an electric field forces them through a medium (matrix) towards the anode.
The most commonly used matrix today is agarose, a natural polymer extracted from sea weeds.
Fragments separate (resolve) according to their size, through the sieving effect of the agarose gel.
★ Exam imp The smaller the fragment, the farther it moves.

Load the DNA samples into the wells at one end of the agarose gel.
Apply an electric field: the DNA moves towards the anode and separates by size.
Stain the DNA with ethidium bromide.
Expose the gel to UV radiation: bright orange-coloured bands of DNA appear.
Cut the separated bands out of the agarose gel.
Extract the DNA from the gel piece: this step is called elution.
Use the purified DNA fragments to construct recombinant DNA by joining them with cloning vectors.

Pure DNA fragments cannot be seen in visible light without staining.

Test yourself: hide labels

Figure 3: A typical agarose gel electrophoresis showing the migration of undigested DNA (lane 1) and digested sets of DNA fragments (lanes 2 to 4). Smaller fragments move farther from the wells.

Memory Trick Run to red: DNA is negative, so it runs to the positive electrode (anode, the red lead). Small fragments run farthest.

Reading a gel: the wells mark where the sample was loaded, at the end away from the anode (the cathode end).
Undigested DNA (lane 1 in Figure 3) stays as one large band near its well.
Digested sets of fragments (lanes 2 to 4) show several bands: the largest near the wells and the smallest farthest away.

★ Exam imp Elution: cutting the separated DNA bands out of the agarose gel and extracting the DNA from the gel piece.

Key idea

Gel electrophoresis sorts DNA fragments by size; ethidium bromide and UV show them, and elution recovers the one you need.

4. Cloning Vectors

4.1 Plasmids, bacteriophages and copy number

Plasmids and bacteriophages can replicate within bacterial cells independently of the control of chromosomal DNA.
Bacteriophages are present in high numbers per cell, so they have very high copy numbers of their genome within the bacterial cells.
★ Exam imp Some plasmids have only one or two copies per cell, while others have 15-100 copies per cell; their numbers can go even higher.
An alien DNA linked with a bacteriophage or plasmid DNA is multiplied to a number equal to the copy number of the plasmid or bacteriophage.
Present-day vectors are engineered for easy linking of foreign DNA and selection of recombinants from non-recombinants.

4.2 Features needed to facilitate cloning into a vector

(i) Origin of replication (ori)

The sequence from where replication starts; any piece of DNA linked to it can be made to replicate within the host cells.
★ Exam imp The ori also controls the copy number of the linked DNA.
To recover many copies of the target DNA, clone it in a vector whose ori supports a high copy number.

(ii) Selectable marker

Besides an ori, the vector needs a selectable marker.
It helps to identify and eliminate non-transformants and selectively permits the growth of transformants.
Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are useful selectable markers for E. coli.
Normal E. coli cells do not carry resistance to any of these antibiotics.

Transformation: the procedure through which a piece of DNA is introduced into a host bacterium. Cells that take it up are transformants; the rest are non-transformants.

Memory Trick ACT-K: Ampicillin, Chloramphenicol, Tetracycline, Kanamycin, the four antibiotic-resistance markers for E. coli.

(iii) Cloning sites

To link the alien DNA, the vector needs very few, preferably single, recognition sites for the commonly used restriction enzymes.
More than one recognition site within the vector would give several fragments, which complicates gene cloning.
The alien DNA is ligated at a restriction site present in one of the two antibiotic resistance genes.

Test yourself: hide labels

Figure 4: E. coli cloning vector pBR322, showing restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I, Pvu I), ori and the antibiotic resistance genes amp^R and tet^R. rop codes for the proteins involved in the replication of the plasmid.

Part of pBR322	What it is or where it lies
ori	Origin of replication
amp^R	Ampicillin resistance gene; contains the Pst I and Pvu I sites
tet^R	Tetracycline resistance gene; contains the BamH I and Sal I sites
rop	Codes for the proteins involved in the replication of the plasmid
EcoR I, Cla I, Hind III	Restriction sites close together, near the start of tet^R
Pvu II	Restriction site near rop, outside both resistance genes

Memory Trick B and S in tet, two Ps in amp: BamH I and Sal I sit in tet^R; Pst I and Pvu I sit in amp^R. Pvu II is the odd P, outside both genes.

Quick Recall: tap to check

Name the two restriction sites of pBR322 that lie in the tetracycline resistance gene.

BamH I and Sal I.

Name the two restriction sites of pBR322 that lie in the ampicillin resistance gene.

Pst I and Pvu I.

What does rop code for?

The proteins involved in the replication of the plasmid.

4.3 Selecting recombinants with two antibiotics

Example: foreign DNA is ligated at the BamH I site of the tetracycline resistance gene in pBR322.

The recombinant plasmids lose tetracycline resistance, because the foreign DNA is inserted into tet^R.
Plate the transformants on an ampicillin-containing medium: all transformants grow, since amp^R is intact.
Transfer the colonies to a tetracycline-containing medium.
Recombinants grow on ampicillin but not on tetracycline.
Non-recombinants grow on both antibiotics.

Cell	Ampicillin medium	Tetracycline medium
Non-transformant (no plasmid)	No growth	No growth
Transformant, non-recombinant (plasmid without insert)	Grows	Grows
Transformant, recombinant (insert in tet^R)	Grows	No growth

One antibiotic resistance gene helps select the transformants.
The other gets 'inactivated due to insertion' of the alien DNA and helps select the recombinants.

4.4 Insertional inactivation: blue and colourless colonies

Selecting recombinants by inactivating an antibiotic resistance gene is cumbersome: it needs plating on two plates with different antibiotics.
So alternative selectable markers were developed. They tell recombinants from non-recombinants by the ability to produce colour with a chromogenic substrate.
The foreign DNA is inserted within the coding sequence of the enzyme $β$ -galactosidase, which inactivates the gene for this enzyme.

★ Very important Insertional inactivation: inactivation of a gene because foreign DNA is inserted within its coding sequence, as with the

β

-galactosidase gene.

Blue colonies

The plasmid has no insert; $β$ -galactosidase works and acts on the chromogenic substrate. These are non-recombinants.

Colourless colonies

The insert has inactivated the $β$ -galactosidase gene, so no colour forms. These are recombinant colonies.

Memory Trick Blue = Blank: a blue colony has a blank plasmid (no insert); a colourless colony carries the insert.

NEET Focus

Two selection logics, often mixed up. With antibiotics, the recombinant is the colony that fails to grow on the second antibiotic (tetracycline for an insert at BamH I). With $β$ -galactosidase, the recombinant is the colony that fails to make colour. In both, the recombinant is the one that has lost a function.

Tips and Tricks Transformant or recombinant? Every recombinant is a transformant, but not every transformant is a recombinant. The first marker (ampicillin) picks out transformants; the inactivated marker (tetracycline or colour) picks out recombinants.

4.5 Vectors for cloning genes in plants and animals

We learnt gene transfer into plants and animals from bacteria and viruses, which have long known how to deliver genes to transform eukaryotic cells.
★ Exam imp Agrobacterium tumefaciens (also printed as tumifaciens), a pathogen of several dicot plants, delivers a piece of DNA called 'T-DNA'.
T-DNA transforms normal plant cells into a tumour and directs the tumour cells to produce the chemicals the pathogen needs.
Retroviruses in animals can transform normal cells into cancerous cells.
Knowing how pathogens deliver genes has let us turn these tools of pathogens into useful vectors, useful to humans, for delivering genes of interest.

Pathogen	Natural action	Use as a vector
Agrobacterium tumefaciens	Delivers T-DNA; turns plant cells into a tumour	Its tumour inducing (Ti) plasmid is modified into a cloning vector that is no longer pathogenic but still delivers genes of interest into a variety of plants
Retroviruses	Turn normal animal cells into cancerous cells	Disarmed retroviruses deliver desirable genes into animal cells

Once a gene or DNA fragment is ligated into a suitable vector, it is transferred into a bacterial, plant or animal host, where it multiplies.

Key idea

A good vector has an ori, a selectable marker and single recognition sites; an insert that inactivates a marker gene reveals the recombinants.

5. Competent Host (for Transformation with Recombinant DNA)

DNA is a hydrophilic molecule, so it cannot pass through cell membranes.
To force bacteria to take up the plasmid, the cells must first be made 'competent' to take up DNA.

★ Exam imp Treat the bacterial cells with a specific concentration of a divalent cation, such as calcium ( $C a^{2 +}$ ). This increases the efficiency with which DNA enters through pores in the cell wall.
Incubate the cells with the recombinant DNA on ice.
Place them briefly at $42^{\circ} C$ (heat shock).
Put them back on ice. The bacteria now take up the recombinant DNA.

Memory Trick Calcium, cold, hot, cold:

C a^{2 +}

treatment, then ice, a brief heat shock at

42^{\circ} C

and then ice again.

5.1 Other ways to introduce alien DNA

Method	How it works	Suited to
Micro-injection	Recombinant DNA is injected directly into the nucleus	Animal cells
Biolistics or gene gun	Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA	Plants
'Disarmed pathogen' vectors	Allowed to infect the cell, they transfer the recombinant DNA into the host	Plants (Ti plasmid) and animals (retroviruses)

Memory Trick Match the cell to the method: a bacterium is made competent with calcium and heat shock; an animal nucleus takes a needle (micro-injection); a plant cell with a tough wall takes metal bullets (gene gun).

Key idea

Hydrophilic DNA needs help to enter cells: calcium and heat shock for bacteria, micro-injection, gene guns or disarmed pathogens for others.

6. Exam Essentials

Pairs to Match

Item	Matches with
Restriction endonuclease	Cuts DNA at a specific recognition sequence
Hind II	First sequence-specific restriction endonuclease; six base pair recognition sequence
EcoRI	Escherichia coli RY 13; cuts between G and A in GAATTC
Exonuclease	Removes nucleotides from the ends of DNA
Endonuclease	Cuts at specific positions within DNA
DNA ligase	Joins sticky ends
Agarose	Natural polymer from sea weeds; gel matrix
Ethidium bromide with UV	Orange bands of DNA
Elution	Extracting DNA from a cut-out gel band
ori	Starts replication; controls copy number
rop	Proteins for plasmid replication
BamH I and Sal I	Sites in tet^R of pBR322
Pst I and Pvu I	Sites in amp^R of pBR322
$β$ -galactosidase	Insertional inactivation; blue or colourless colonies
Ti plasmid	Agrobacterium tumefaciens; vector for plants

Exceptions

Exonucleases do not cut within DNA; they remove nucleotides from the ends.
The cut is a little away from the centre of the palindrome (as with EcoRI), not at its centre, yet it is between the same two bases on both strands.
Without the same restriction enzyme for vector and source DNA, a recombinant vector cannot normally be made.
Pure DNA is not visible in visible light without staining.
Normal E. coli carries no resistance to ampicillin, chloramphenicol, tetracycline or kanamycin.
Recombinants with an insert at BamH I do not grow on tetracycline.
Blue colonies are non-recombinants; colourless ones are recombinants.
The modified Ti plasmid is no longer pathogenic to plants.
DNA, being hydrophilic, cannot pass through cell membranes on its own.

Numbers to Remember

1963: two enzymes restricting phage growth in E. coli isolated.
Five years after 1963: Hind II isolated and characterised.
6 base pairs: length of the Hind II recognition sequence.
More than 900 restriction enzymes from over 230 bacterial strains.
Plasmid copy number: 1-2 per cell in some, 15-100 per cell in others.
4 antibiotic markers for E. coli: ampicillin, chloramphenicol, tetracycline, kanamycin.
Heat shock at $42^{\circ} C$ .

Examples to Remember

Group	Examples
Restriction enzymes named	Hind II, Hind III, EcoRI, BamH I, Sal I, Pvu I, Pvu II, Pst I, Cla I
Cloning vectors	Plasmids (pBR322), bacteriophages, Ti plasmid, disarmed retroviruses
Selectable marker genes	Resistance to ampicillin, chloramphenicol, tetracycline, kanamycin; $β$ -galactosidase (colour)
Metals in the gene gun	Gold, tungsten
Divalent cation for competence	Calcium

7. Quick Revision

Key tools: restriction enzymes, polymerases, ligases, vectors and a host.
1963: a methylating enzyme and a cutting enzyme (restriction endonuclease) found in E. coli.
Hind II, the first sequence-specific restriction endonuclease, recognises six base pairs; over 900 enzymes now known from over 230 strains.
Naming: genus letter, two species letters, strain, Roman numeral for order of isolation.
Exonucleases trim ends; endonucleases cut within; restriction enzymes are endonucleases.
They inspect DNA, bind the palindromic recognition site and cut both sugar-phosphate backbones.
The off-centre cut leaves sticky ends that hydrogen-bond and help DNA ligase.
Vector and source DNA must be cut with the same enzyme.
Gel electrophoresis: DNA to anode through agarose; small fragments farthest; ethidium bromide plus UV; elution.
Plasmids and phages replicate independently; copy number 1-2 or 15-100 or more.
Vector features: ori (controls copy number), selectable marker, very few (preferably single) recognition sites.
pBR322: insert at BamH I kills tetracycline resistance; recombinants grow on ampicillin only.
$β$ -galactosidase insertional inactivation: blue = no insert, colourless = recombinant.
Ti plasmid of Agrobacterium and disarmed retroviruses carry genes into plants and animals.
Competence: $C a^{2 +}$ , ice, $42^{\circ} C$ , ice; also micro-injection, gene gun, disarmed pathogens.

8. Solved Examples

Solved Example 1

Match List I with List II.
List I: A. BamH I, B. Pst I, C. rop, D. ori
List II: I. Codes for proteins of plasmid replication, II. Site in the ampicillin resistance gene, III. Site in the tetracycline resistance gene, IV. Controls the copy number of linked DNA
Choose the correct answer:
(A) A-III, B-II, C-I, D-IV
(B) A-II, B-III, C-I, D-IV
(C) A-III, B-II, C-IV, D-I
(D) A-I, B-II, C-III, D-IV

Solution:

Answer: (A). BamH I lies in tet^R (III), Pst I in amp^R (II), rop codes for replication proteins (I) and ori controls copy number (IV).

Solved Example 2

Read the statements.
A. In gel electrophoresis, DNA fragments move towards the anode.
B. Larger DNA fragments move farther through the agarose gel.
C. Agarose is a natural polymer extracted from sea weeds.
D. DNA bands are seen after staining with ethidium bromide and exposure to UV light.
E. Elution is the loading of DNA into the wells.
Choose the correct answer:
(A) A, C and D only
(B) A, B and C only
(C) B, D and E only
(D) A, C, D and E only

Solution:

Answer: (A). B is wrong: smaller fragments move farther. E is wrong: elution is extracting DNA from a cut-out gel band.

Solved Example 3

Arrange the steps that make bacterial cells take up recombinant DNA in the correct order.
A. Heat shock at

42^{\circ} C

B. Treatment with calcium ions
C. Incubation with recombinant DNA on ice
D. Return to ice
Choose the correct answer:
(A) C, B, A, D
(B) B, C, A, D
(C) B, A, C, D
(D) A, B, C, D

Solution:

Answer: (B). Cells are made competent with calcium (B), incubated with DNA on ice (C), given a brief heat shock (A) and put back on ice (D).

Solved Example 4

Statement I: Restriction enzymes cut DNA exactly at the centre of the palindrome on both strands.
Statement II: Sticky ends form hydrogen bonds with their complementary cut counterparts.
Choose the correct answer:
(A) Both Statement I and Statement II are correct
(B) Both Statement I and Statement II are incorrect
(C) Statement I is correct but Statement II is incorrect
(D) Statement I is incorrect but Statement II is correct

Solution:

Answer: (D). The cut is a little away from the centre, though between the same two bases on both strands, so Statement I is wrong. Statement II explains the name 'sticky'.

Solved Example 5

Foreign DNA is ligated at the BamH I site of pBR322. Which colonies are the recombinants?
(A) Those growing on both ampicillin and tetracycline
(B) Those growing on ampicillin but not on tetracycline
(C) Those growing on tetracycline but not on ampicillin
(D) Those growing on neither antibiotic

Solution:

Answer: (B). The insert inactivates tet^R, while amp^R stays intact.

Solved Example 6

Which of the following statements is NOT correct?
(A) In EcoRI, the letter R comes from the strain
(B) Exonucleases make cuts at specific positions within DNA
(C) Hind II recognises a six base pair sequence
(D) Restriction enzymes belong to the class of nucleases

Solution:

Answer: (B). Exonucleases remove nucleotides from the ends; endonucleases cut within the DNA.

9. Practice Questions

Practice Questions

Make a chart, with a diagram, showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.Answer: Enzyme: EcoRI. Substrate: double-stranded DNA with the palindrome 5'-GAATTC-3' / 3'-CTTAAG-5'. Site: between G and A on each strand (G^AATTC). Product: two fragments, each with a single-stranded AATT sticky end (see Figure 1).
Are enzymes bigger or is DNA bigger in molecular size? How do you know?Answer: DNA is far bigger. A restriction enzyme 'inspects' along the length of a DNA molecule and binds only at a short six base pair site, and a single DNA molecule can be cut into many fragments, so the DNA is many times longer than the enzyme.
Do eukaryotic cells have restriction endonucleases? Justify your answer.Answer: No. Restriction endonucleases are part of a bacterial defence against bacteriophages. They work with a methylating enzyme (the pair found in 1963): methyl groups mark the bacterium's own DNA, while the endonuclease cuts the unmarked, invading phage DNA. Eukaryotic cells do not have this restriction system.
Collect five examples of palindromic DNA sequences, and create one by following the base-pair rules.Answer: GAATTC (EcoRI), GGATCC (BamH I), AAGCTT (Hind III), GTCGAC (Sal I), CTGCAG (Pst I). To create one, write any three bases, then add the complements of these three in reverse order: GAT + ATC = GATATC.
How can a reporter enzyme be used to monitor transformation of host cells by foreign DNA, in addition to a selectable marker?Answer: The vector carries the gene of a reporter enzyme such as $β$ -galactosidase. Cells with the vector make the enzyme, which turns a chromogenic substrate blue. If the foreign DNA is inserted into this gene, it is inactivated and the colonies stay colourless, which marks the recombinants.
Describe briefly: (a) origin of replication; (b) restriction enzymes and DNA.Answer: (a) The sequence where replication starts; DNA linked to it replicates in the host, and it controls the copy number. (b) Restriction endonucleases inspect DNA, bind a specific palindromic recognition sequence and cut both strands at specific points in the sugar-phosphate backbone, leaving sticky ends.
Distinguish between: (a) plasmid DNA and chromosomal DNA; (b) exonuclease and endonuclease.Answer: (a) Plasmid DNA is small, circular and extra-chromosomal, replicates independently and may have many copies; chromosomal DNA is the main genome, replicating once per cell cycle. (b) An exonuclease removes nucleotides from DNA ends; an endonuclease cuts at specific positions within the DNA.
Match List I with List II.
List I: A. Micro-injection, B. Biolistics, C. Calcium ions, D. Ti plasmid
List II: I. Makes bacterial cells competent, II. Disarmed pathogen vector for plants, III. Gold or tungsten particles coated with DNA, IV. DNA injected into the nucleus of an animal cell
Choose the correct answer:
(A) A-IV, B-III, C-I, D-II
(B) A-III, B-IV, C-I, D-II
(C) A-IV, B-III, C-II, D-I
(D) A-I, B-III, C-IV, D-IIAnswer: (A). Micro-injection into an animal nucleus; biolistics uses coated gold or tungsten; calcium makes cells competent; the Ti plasmid is a disarmed vector for plants.
Read the statements.
A. Hind II was the first sequence-specific restriction endonuclease to be isolated.
B. Over 900 restriction enzymes have been isolated from over 230 bacterial strains.
C. Roman numerals in an enzyme's name show the species.
D. Each restriction endonuclease recognises a palindromic sequence.
Choose the correct answer:
(A) A, B and D only
(B) A, B and C only
(C) B, C and D only
(D) A and C onlyAnswer: (A). C is wrong: Roman numerals show the order of isolation from the strain.
Arrange the steps of recovering a DNA fragment from a gel in order.
A. Elution
B. Staining with ethidium bromide and exposure to UV
C. Separation by electrophoresis
D. Cutting out the band
(A) C, B, D, A
(B) B, C, D, A
(C) C, D, B, A
(D) C, B, A, DAnswer: (A). Separate, stain and view, cut out the band, then elute the DNA.
Statement I: Selection using two antibiotic plates is cumbersome.
Statement II: In $β$ -galactosidase selection, colonies with an insert turn blue.
Choose the correct answer:
(A) Both Statement I and Statement II are correct
(B) Both Statement I and Statement II are incorrect
(C) Statement I is correct but Statement II is incorrect
(D) Statement I is incorrect but Statement II is correctAnswer: (C). Statement II is wrong: colonies with an insert stay colourless; blue colonies have no insert.
Which of the following is NOT a feature required in a cloning vector?
(A) Origin of replication
(B) Selectable marker
(C) Many recognition sites for each common restriction enzyme
(D) Ability to replicate within the host cellAnswer: (C). A vector needs very few, preferably single, recognition sites; many sites would give several fragments.

Common Mistakes to Avoid

Watch out

Saying DNA moves to the cathode in gel electrophoresis. Correct: it is negatively charged, so it moves to the anode.
Writing that larger fragments move farther. Correct: smaller fragments move farther.
Reading the R in EcoRI as 'restriction'. Correct: R is the strain, RY 13.
Calling exonucleases site-specific cutters. Correct: endonucleases cut within DNA; exonucleases trim the ends.
Taking blue colonies as recombinants. Correct: blue colonies have no insert; colourless colonies are recombinants.
Placing Pst I in the tetracycline resistance gene. Correct: Pst I and Pvu I are in amp^R; BamH I and Sal I are in tet^R.
Saying ligase cuts DNA. Correct: restriction enzymes cut; ligase joins the cut ends.
Heat shock at $37^{\circ} C$ . Correct: $42^{\circ} C$ , between two periods on ice.

Frequently Asked Questions

What are the tools of recombinant DNA technology?

The key tools are restriction enzymes, which cut DNA at specific sites; polymerase enzymes, which copy DNA; ligases, which join DNA ends; vectors such as plasmids and bacteriophages, which carry DNA into cells; and a competent host organism, usually Escherichia coli, which multiplies the recombinant DNA.

How are restriction enzymes named?

The first letter comes from the genus and the next two from the species of the bacterium; a further letter gives the strain, and a Roman numeral gives the order of isolation. In EcoRI, E is Escherichia, co is coli, R is strain RY 13 and I means it was the first enzyme isolated from that strain.

What is a palindromic sequence in DNA?

It is a sequence of base pairs that reads the same on both strands when both are read in the same direction. For example, GAATTC on one strand pairs with CTTAAG, which also reads GAATTC from its $5^{'}$ end. Each restriction endonuclease recognises such a palindromic site.

What are sticky ends and why are they important?

Restriction enzymes cut the two strands a little away from the centre of the palindrome, leaving short single-stranded overhangs called sticky ends. They form hydrogen bonds with complementary ends, so DNA cut by the same enzyme from different sources can be joined by DNA ligase to make recombinant DNA.

How does gel electrophoresis separate DNA fragments?

DNA is negatively charged, so in an electric field it moves through an agarose gel towards the anode. The gel acts as a sieve, so smaller fragments move farther. Bands are seen after staining with ethidium bromide under UV light, then cut out and recovered by elution.

What features must a cloning vector have?

A cloning vector needs an origin of replication, which starts replication and controls copy number; a selectable marker, such as an antibiotic resistance gene, to pick out transformants; and very few, preferably single, recognition sites for common restriction enzymes, where foreign DNA can be inserted.

How are recombinants selected using pBR322?

If foreign DNA is inserted at the BamH I site, the tetracycline resistance gene is inactivated. Transformants are first grown on ampicillin. Those that also grow on tetracycline are non-recombinants; those that fail to grow on tetracycline carry the insert; these are the recombinants. NEET often asks this logic.

How is a bacterial cell made competent to take up DNA?

As described in NCERT, bacterial cells are treated with a specific concentration of a divalent cation such as calcium. They are incubated with recombinant DNA on ice, given a brief heat shock at $42^{\circ} C$ and returned to ice. DNA, being hydrophilic, cannot otherwise pass through the cell membrane.

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