Tools of Genetic Engineering to Make Genome Modifications

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Tools of Genetic Engineering to Make Genome Modifications

Introduction

The field of genetic engineering includes many techniques and depends on a group of tools to manipulate DNA. Two of the most useful are restriction enzymes and cloning vectors.

Restriction Enzymes

One of the first demonstrations of genetic engineering involved cutting open the circular DNA molecule from simian virus-40 (SV40) and then inserting (splicing) it into a bacterial chromosome. In doing so, a recombinant DNA molecule, a DNA molecule containing DNA segments from two or more organisms, was created.

To facilitate the process, enzymes called restriction endonucleases can be used. These enzymes act like molecular scissors to recognize and cut specific short stretches of nucleotides in DNA. The sequences recognized by the enzymes are called palindromes because the bases have the same sequence on both DNA strands when read in the 5’ to 3’ direction.

Examples of Restriction Endonuclease Recognition Sequences

Organism Restriction Enzyme Recognition Sequence
Escherichia coli EcoRI G ↓ AATTC
CTTAA ↑ G
Streptomyces albus SalI G ↓ TCGAC
CAGCT ↑ G
Haemophilus influenzae HindIII A ↓ AGCTT
TTCGA ↑ A
Bacillus amyloliquefaciens BamHI G ↓ GATCmC
CCmTAG ↑ G
Providencia stuartii PstI CTGCA ↓ G
G ↑ ACGTC
Enzyme designations are derived from the species from which they were isolated. For example, the restriction
enzyme EcoRI stands for Escherichia coli Restriction enzyme I.
Arrows indicate where the restriction enzyme cuts the two strands of the recognition sequence; Cm = methylcytosine

Today, there is a vast array of restriction enzymes that have been isolated from prokaryotic organisms. Each restriction enzyme recognizes a specific nucleotide sequence. Importantly, many of these enzymes leave the DNA with single-stranded extensions, called “sticky ends,” that can easily attach to complementary ends protruding from another fragment of DNA. To seal these complementary DNA segments, DNA ligase is used.

Putting this all together, microbiologists can take a plasmid from a bacterial species such as E. coli and open it with a restriction enzyme. Then, they can insert a segment of foreign DNA into the plasmid and seal the segment by using DNA ligase.

Construction of a Recombinant DNA Molecule
In this construction, two unrelated plasmids (loops of DNA) are united to form a single plasmid representing a recombinant DNA molecule.

Cloning Vectors

One of the goals of genetic engineering is to insert a useful, foreign gene into another cell that will then produce the protein product of that gene. To carry this gene to the target recipient cell, a cloning vector is used. This vector can be a transposon, viral genes, or a bacterial plasmid.

The best way to understand how the vector works and to see how genetic engineering operates is to follow the steps that have been used to clone a human gene into E. coli. Today, more than 18 million people in the United States have diabetes, a group of diseases resulting from abnormally high blood glucose levels.

There are more than 800,000 cases of insulin-dependent diabetes (also called juvenile or type I diabetes), requiring these diabetics to receive regular injections of insulin to control their blood glucose level. Before 1982, insulin was extracted and purified from the pancreas of cattle and pigs or even cadavers.

However, this posed a problem because the animal insulin protein is not identical in amino acid sequence to the human insulin protein, and such an animal protein can trigger allergic reactions in the diabetic individual. In addition, the animals used to extract the insulin could contain unknown disease causing viruses that would be isolated along with the insulin protein. The solution was to produce human insulin by using genetic engineering. Such method—by cloning the human gene for insulin into bacterial cells, which then act as tiny factories to synthesize and secrete human insulin.

Reference and Sources

  • https://quizlet.com/271022533/micro-chapter-8-flash-cards/
  • https://opentextbc.ca/biology/chapter/10-1-cloning-and-genetic-engineering/
  • https://diabetestalk.net/diabetes/nursing-interventions-for-diabetes-type-2
  • http://www.madehow.com/Volume-7/Insulin.html
  • https://www.sciencedirect.com/science/article/pii/B9780128200070000222
  • https://www.chemeurope.com/en/encyclopedia/Insulin.html
  • https://www.biologydiscussion.com/dna/recombinant-dna-technology/insertion-of-a-foreign-dna-fragment-intoa-vector/37319
  • https://en.wikipedia.org/wiki/Genetic_engineering_techniques

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