Restriction Enzymes, Cloning Vectors & DNA Profiling – Review

18 question-and-answer flashcards summarizing sticky-end recognition, plasmid features, cloning vectors, mapping logic, and DNA profiling methods covered in the lecture.

What type of molecular tool produces “sticky ends” in DNA?

Restriction (endonuclease) enzymes that cut at specific recognition sequences.

How do you determine whether a sticky end is a 5′ or 3′ overhang?

Look at the single-stranded protrusion; the overhang is named for the direction (5′→ or 3′→) that the protruding strand points toward.

If the single-stranded overhang points toward the 5′ end of the strand, what is it called?

A 5′ overhang (5-prime overhang).

If the single-stranded overhang points toward the 3′ end of the strand, what is it called?

A 3′ overhang (3-prime overhang).

Which restriction enzyme recognizes the sequence GAATTC?

EcoR I.

A circular plasmid has one EcoR I cut site. After digestion with EcoR I, how many fragments appear and what is their form?

One linear fragment equal to the full plasmid size.

Name four commonly used cloning vectors in recombinant DNA work.

Plasmids, bacteriophages, cosmids, BACs (bacterial artificial chromosomes) and YACs (yeast artificial chromosomes).

List at least four key features that must appear in a standard cloning plasmid.

1) Origin of replication (ori) 2) Antibiotic-resistance gene 3) Multiple cloning site / restriction enzyme sites 4) Selectable marker (e.g., lacZ for blue-white screening) 5) Overall plasmid size information.

Which gene is exploited for blue-white screening in plasmid cloning?

The lacZ gene encoding β-galactosidase.

In plasmid mapping, what is the relationship between number of cut sites and number of fragments for a circular DNA?

Fragments = #Cut Sites when circular DNA is digested; one extra fragment appears only if the molecule is linear to start with.

What happens to a 20-kb circular plasmid when cut once with Hind III?

It converts to one 20-kb linear fragment.

Give the full names for the three DNA profiling techniques RFLP, VNTR and STR.

RFLP – Restriction Fragment Length Polymorphism; VNTR – Variable Number Tandem Repeat; STR – Short Tandem Repeat analysis.

Which two profiling techniques rely primarily on restriction enzymes and gel electrophoresis?

RFLP and VNTR analyses.

Which profiling technique requires the LEAST starting DNA and is fastest, and why?

STR analysis; it uses PCR with fluorescent primers, so only trace DNA is needed and results are rapid.

Why does RFLP analysis demand relatively large quantities of DNA?

Because it analyzes many restriction fragments across the genome without amplification, so sufficient starting material is needed to visualize bands after electrophoresis.

What plasmid feature contains a cluster of different restriction sites to facilitate insertion of foreign DNA?

The multiple cloning site (MCS), also called the polylinker.

What is the role of the origin of replication (ori) in a plasmid vector?

It ensures the plasmid is replicated within the host cell, allowing propagation of cloned DNA.

During double-digest mapping, why is it helpful to first diagram the cuts made by each enzyme separately?

It clarifies the individual fragment sizes and locations, making it easier to deduce where sites overlap when both enzymes act together.