Restriction enzymes and DNA fingerprinting

DNA fingerprinting is used in the legal world to determine parentage, genealogy, or other genetic relationships, or to implicate suspects in law enforcement investigations involving violent crimes. Fragments produced by various restriction enzymes from a number of different loci can be used to identify individuals with the accuracy of a fingerprint. Therefore, this technique is called “Fingerprinting”.

Which “one” of the following statements is incorrect about restriction enzymes?

A. They are site specific endonucleases

B.  The restriction sites are palindrome in nature

C. All restriction enzymes are of bacterial origin

D. They always produce complementary single stranded overhangs

E. They are also called “molecular scissors”.

The correct answer is-D.

DNA fingerprinting works on the basis that each individual’s DNA structure or genetic make-up is unique and therefore cannot be forged, faked or altered in any way. Just like normal fingerprints taken from a suspect are so unique that only in the case of identical twins, could they be the same. Statistically there is a one in sixty-four billion chance that any two unrelated individuals would have comparable DNA: comparable DNA is DNA that has certain attributes similar to that of another person but is not identical.

One of the most common DNA fingerprinting procedures is RFLP: Restriction Fragment Length Polymorphism. The procedure of RFLP focuses on repetitious sequences of DNA ‘bases, ‘Variable numbers of tandemly repeated (VNTR) units, which vary greatly from individual to individual. These differences in DNA sequence can result in variations of restriction sites and thus in the length of restriction fragments (Figure-1). An inherited difference in the pattern of restriction (e.g., a DNA variation occurring in more than 1% of the general population) is known as a restriction fragment length polymorphism, or RFLP.

The VNTRs can be inherited, in which case they are useful in establishing genetic association with a disease in a family or they can be unique to an individual and thus serve as a molecular fingerprint of that person.

RFLP

Figure-1- Variations in the nucleotide sequence at the restriction site produce different sized restriction fragments.

The differences in restriction sites form the basis for paternity test (Figure-2)

Paternity test

Figure-2- Paternity test

Restriction endonucleases

  • These enzymes were called restriction enzymes because their presence in a given bacterium restricted the growth of certain bacterial viruses called bacteriophages.
  • Restriction enzymes cut DNA of any source into short pieces in a sequence-specific manner
  • The specific site is called “Restriction site” which is 4-7 base pair long, and is palindrome in nature
  • The fragments of DNA obtained after the action of restriction enzymes are called “Restriction fragments”
  • Based on their function, Restriction enzymes are also called “Molecular scissors”
  • There are three types of restriction endonucleases designated as Type I, II and III.
  • Type I and III require ATP
  • Type II do not require ATP and cleave the DNA  within the restriction site itself
  • Type II restriction endonucleases are the most widely used enzymes for recombinant DNA technology
  • Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamHI is from Bacillus amyloliquefaciens
  • DNA cuts result in blunt ends (e.g., HpaI) or overlapping (sticky) ends (e.g., BamHI) depending on the mechanism used by the enzyme (Figure-3).

Sticky and blunt ends

If an enzyme makes a staggered cut, the sticky ends are produced

  • Blunt ends are produced by direct cut
  • Sticky means the overhangs on the fragments are single stranded, and are complementary to each other, if brought closer to each other, can reanneal (Figure-3).
  • Blunt ends have no overhangs, so they cannot hydrogen bond with each other.
  • Sticky-end ligation is technically easy, but Sticky ends of a vector may reconnect with themselves, with no net gain of DNA.
  • Sticky ends of fragments can also anneal, so that tandem heterogeneous inserts form.
  • Also, sticky-end sites may not be available or in a convenient position.
  • To circumvent the problem with sticky ends, an enzyme that generates blunt ends is used,
  • New ends are added using the enzyme terminal transferase.
  •  If poly d(G) is added to the 3′ ends of the vector and poly d(C) is added to the 3′ ends of the foreign DNA,
  • The two molecules can only anneal to each other, thus circumventing the problems listed above.

 

Restriction fragments

Figure-3- Sticky and blunt ends of restriction fragments

As regards other options

All statements correctly describe about restriction endonucleases except for the complementary single stranded overhangs(Sticky ends) which are not always produced. Blunt ends can also be produced,  depending upon the nature of the enzyme and the type of cut involved.

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