Cloning Vectors

A selectable marker helps identify transformed cells. Antibiotic resistance is a common example.

A cloning site provides a defined place for inserting DNA into a vector. Remember it as the insert entry region.

A small vector is easier to isolate and introduce into a host. Therefore small size is a practical advantage.

Plasmids are small independent circular DNA molecules. They are very useful as cloning vectors.

The governing principle is plasmid copy number. Copy number means the average number of vector molecules maintained in one host cell. If a compatible plasmid has a high copy number, each transformed cell can contain many copies of the vector and, consequently, many copies of the inserted DNA. After growing a large population of such cells, the total recovered DNA is generally higher, provided that the insert remains stable and the host tolerates the plasmid. Option C is correct. A low copy number usually gives less DNA, absence of ori prevents normal plasmid replication, and absence of a selectable marker affects identification rather than directly increasing yield.

The governing concept is biological gene delivery by a plant-associated vector. The Ti plasmid, or tumour-inducing plasmid, naturally occurs in the bacterium Agrobacterium tumefaciens. A defined region of this plasmid, the T-DNA, can be transferred into a plant cell and integrated into the plant genome. Biotechnology researchers modify the Ti plasmid by removing disease-causing genes while retaining its transfer system, so it can carry a useful gene into plant cells. Option C is correct. pUC plasmids are common laboratory cloning plasmids, BACs are designed for large DNA fragments, and pepsin is a digestive enzyme rather than a vector.

Agrobacterium has the ability to transfer T-DNA into plants. This makes it useful in plant transformation.

The direct answer is B: a multiple cloning site contains many restriction-enzyme recognition sites. A restriction enzyme cuts DNA at its own specific sequence. In an MCS, several different recognition sequences are placed close together in a short region. This gives the researcher choices: an enzyme can be selected that cuts the insert and vector suitably, sometimes producing compatible ends and useful orientation. Option B is correct. Option A is wrong because an MCS is a DNA sequence region, not a protein. Option C is wrong because it does not contain only water; it is made of DNA bases. Option D is wrong because an MCS is specifically part of the vector DNA, not a DNA-free region. The sites should be arranged so that unwanted cutting elsewhere in the vector is avoided. Memory cue: multiple cloning site means many enzyme doors for one insert.

pBR322 is a classic plasmid cloning vector. It contains parts such as ori and selectable markers.

In transformation external DNA or recombinant vector is introduced into a host cell. It is an important step in cloning workflow.

The direct answer is A: a recombinant vector. A cloning vector is a DNA molecule designed to carry another DNA fragment. First, foreign DNA is selected as the insert. It is placed into a suitable site of the vector, and ligase joins the two DNA parts. Because the final molecule contains DNA from two different sources, it is called recombinant DNA; when the carrier is the vector, it is a recombinant vector. A is correct. B, pure water, contains no joined genetic material. C, chlorophyll, is a pigment involved in photosynthesis and is not produced simply by joining DNA to a vector. D, a starch grain, is a storage structure or starch form and has no connection with vector construction. The recombinant vector can later be introduced into a host and copied if it has suitable features such as an ori. Memory cue: “foreign DNA + vector = recombinant vector.”

A cloning site provides the designed location for adding insert DNA. Without it insertion will not be controlled.

The governing concept is selection of transformed host cells. A selectable marker is a gene carried by the vector that gives cells containing the vector a detectable survival or growth advantage under a defined condition. For example, an antibiotic-resistance marker can allow transformed bacterial cells to grow on medium containing that antibiotic, whereas cells without the vector are inhibited. Option A is correct because the marker distinguishes vector-containing cells from non-transformed cells. A circular shape may be common in plasmids but is not a selection mechanism. Gel colour and a fat layer neither identify transformants nor control their growth.

A host cell provides cellular machinery to replicate the vector. This makes copies of desired DNA.

Escherichia coli is a common host for plasmid cloning. It grows fast and is easy to handle in labs.

BAC stands for bacterial artificial chromosome. It is engineered from elements of bacterial plasmids and chromosomes so that it can maintain a comparatively large foreign DNA fragment in a bacterial host. BACs are useful in genomic libraries, physical mapping, and the study or sequencing of long genomic regions because they are generally maintained at low copy number, which can improve stability of large inserts. Option A is correct. Amylase and lipase are digestive enzymes, while insulin is a peptide hormone; none of these molecules is a DNA cloning vector. The defining clue is the ability to carry a large insert, not enzymatic or hormonal activity.

YAC stands for Yeast Artificial Chromosome. It is a cloning vector that functions in yeast and is used to carry and clone very large DNA fragments. ‘Young animal cell’ merely describes a cell and is not a cloning vector. Exam tip: remember YAC as Y = Yeast and AC = Artificial Chromosome.

A bacteriophage is a virus that infects bacteria. Its modified forms can become cloning vectors.

A cosmid is a plasmid-like vector with phage cos sites. It can be useful for larger DNA inserts.

A promoter is a regulatory DNA sequence where RNA polymerase and associated factors begin transcription. It is especially necessary when the purpose of the vector is not merely to replicate an inserted DNA fragment but to express that gene and produce its RNA or protein in the host. The promoter must usually be recognized by the host’s transcription machinery and placed in a suitable orientation and position relative to the gene. Option A is correct. Water requirement, vector removal, and burning a host do not require transcriptional regulation and are unrelated to promoter function.

The aim of a cloning vector is to increase DNA copies. An expression vector can be designed to produce gene product.

An insert can disrupt the lacZ gene. This can produce a white colony indicating a recombinant clone.

An insert can enter a marker gene and stop its activity. This is called insertional inactivation.

Antibiotic selection chooses vector-containing cells. Screening or confirmation tells whether insert is present.

Restriction digestion can show an expected DNA fragment pattern. This indicates insert presence.