UNIT 2:  MOLECULAR GENETICS

 

A.  The Tools and Techniques of Biotechnology  

Just as carpenters, mechanics, surgeons, and dentists require tools to do their job, molecular biologists use tools to assist them in completing projects like investigating genetic disorders, altering the genetic makeup of organisms so they can produce products such as insulin, or analyzing DNA evidence in criminal investigations.  The difference between the tools of molecular biologists versus those of other professions is that the tools for molecular biologists are often living organisms or biological (organic) molecules.  That means that the tools are dynamic and are sensitive to environmental influences.  Molecular biologists can cut, join, and replicate sections of DNA at will.  They can remove sections from one DNA molecule and transfer them to another, forming recombinant DNA.

 Restriction Endonucleases

·         restriction endonucleases are also known as restriction enzymes  (r.e.) – molecular scissors that cut double-stranded DNA at a specific base-pair sequence

·         each r.e. notices a particular base sequence as its recognition site where it does the “cutting”

·         most recognition sites are about four to eight base pairs long and are characterized by palindromic sequences (see Table 1, p. 279)

·         Figure 1, p. 278 shows how the r.e. EcoRI works to cleave a DNA molecule

·         r.e.s bind to their recognition sites and disrupts the phosphodiester bond between nucleotides, via hydrolysis, which in turn, disrupts the H-bonds between nitrogenous bases

·         EcoRI produces fragments with sticky ends – both fragments have DNA nucleotides that lack their respective complementary bases, producing overhangs

·         for an animation of EcoRI action, click on http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/enuc.html

·         another r.e. called SmaI, produces blunt ends – both ends of each fragment are fully base-paired

·         Table 1, p. 279 shows the kinds of fragments that can result from the work of r.e.s

·         sticky-end fragments are generally more useful because they can be easily joined with other sticky end fragments for DNA incorporation and recombinant construction

·         the longer the base pair sequence of any recognition site is the less likely it will occur

·         for example, the probability of finding a six-base-pair sequence, such as that of EcoRI, is 4x4x4x4x4x4, once in every 4096 nucleotides, whereas the probability of finding a two-base-pair sequence, such as that of AluI, is 4x4, once in every 16 nucleotides

·         the decrease in occurrence of longer recognition sites results in fewer cuts – this ensures that an entire gene remains in tact

·         the increase in occurrence of shorter recognition sites results in more cuts – this makes shorter fragments

·         both are useful, however, r.e.s that cleave at six-base-pair recognition sites result in a frequency of cleavage that can be used for many applications

·         r.e.s in bacteria are used as a bacteria’s defence mechanism – the bacteriophages DNA is cleaved by the host bacteria’s r.e.s making it inactive and “non-transcribable”

·         r.e.s are named according to the bacteria from which they originate

·         for example, BamHI is named as follows:  B represents the genus Bacillus, am represents the species amyloliquefaciens, H represents the strain, and I means that it was the first endonuclease isolated from this strain

·         another example is HindII is named as follows:  H represents the genus Haemophilus, in represents the species influenzae, d represents the strain Rd, and II means that it was the second endonuclease isolated from this strain

·         in the 1960s, Hamilton Smith, of John Hopkins University, noticed that the DNA that was removed from one bacterial strain of E. coli, and inserted into another strain of E. coli, did not survive but instead was cut into pieces

·         since then, more than 2500 r.e.s with specificity for about 200 different target sites have been isolated from prokaryotic organisms

 Homework:  1-5, p. 281

 Methylases 

 ·         r.e.s must be able to recognize the difference between foreign DNA and their own

·         methylases are specific enzymes found in prokaryotes and eukaryotes

·         in prokaryotes, they modify the recognition site of a respective restriction endonuclease by placing a methyl group on one of the bases, preventing the restriction endonuclease from cutting the DNA into fragments

·         for example, EcoRI methylase, adds a methyl group to the second adenine nucleotide in the EcoRI recognition site (see Figure 3, p. 281) preventing EcoRI from cutting the DNA

·         foreign DNA is not methylated, or “protected”, rendering it defenceless against the bacterium’s restriction enzymes

·         these are important tools for a molecular biologist when working with prokaryotic organisms – they help protect a gene fragment from being cleaved in an undesired location

 DNA Ligase

 ·         genes that are cut out from DNA sources must be rejoined back into a sequence of existing DNA

·         DNA ligase is the enzyme that is used as the tool to join sticky-ended fragments of DNA together – using a condensation reaction, DNA ligase drives out a molecule of water and reforms the phosphodiester bond of the backbone of the DNA

·         sticky DNA fragments are easier to rejoin than blunt ended-DNA fragments

·         T4 DNA ligase, an enzyme that originated from the T4 bacteriophage, is used to join blunt ends together (see Figure 4, p. 282)

·         the following diagram shows how EcoRI and DNA ligase work on a fragment of DNA:  http://www.mun.ca/biology/scarr/Fg15_02.gif

 Homework:    6-10, p. 282

 Gel Electrophoresis 

·         once the desired gene has excised from its source DNA, it must be separated from the remaining unwanted fragments

·         DNA fragments can be separated using a process called gel electrophoresis – a process that works based on the physical and chemical properties of the DNA molecule

·         DNA is a negatively charged molecule – each nucleotide possesses a phosphate group that has a net charge of – 1

·         the difference in molecular mass between each nucleotide is negligible

·         this means that all nucleotides have the same charge-to-mass-ratio

·         two different strands of DNA of the same length carry the same charge-to-mass ratio

·         two strands of DNA of different lengths possess a different charge-to-mass ratio

·         gel electrophoresis is like a “molecular sieve” – DNA fragments, of various sizes, that have been created by r.e. digestion, will be separated from one another

·         the fragments will migrate through the gel at a rate that is inversely proportional to the logarithm of their size – the shorter the fragment is, the faster it will travel because of its ability to navigate through the pores in the gel more easily than a large fragment can

·         shorter fragments navigate further distances down the gel, whereas longer fragments get “snagged” further up the gel (see Figure 6, p. 283)

·         here’s how it works:

• a solution containing different sized fragments is placed in a well depression at the top of the gel
• the gel consists of a jelly-like square or rectangular slab of buffer-containing electrolytes and agarose or polyacrylamide
• the DNA fragments are mixed with a loading dye containing glycerol
• glycerol is used because it is a heavy substance so it helps the DNA solution to sink down into the well
• the loading dye is used to make the DNA solution visible in the gel
• using DC electricity, a negative charge is placed at one end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel
• the electrolyte solution conveys the current through the gel
• the negatively-charged DNA migrates to the opposite side (the positive electrode), with the shorter fragments migrating further down the gel
• small molecules found within the loading dye migrate ahead of all the DNA fragments – before they reach the bottom, the electricity is turned off
• once the process is over, the DNA fragments are made visible by staining the gel (see Figure 7, p. 284)
• the set of fragments that result with a particular restriction enzyme produces a banding pattern characteristic for that DNA
• a commonly used stain is ethidium bromide (see Figure 8, p. 284) because it fluoresces under UV light
• ethidium bromide inserts itself among the nucleotides, which is the reason that the fragments can be seen
• the size of the fragments can be measured by comparing them with a molecular marker (a sample containing fragments of known size run through the same gel)
• the log of the known fragment sizes is plotted versus the distance traveled in the gel, resulting in a graph that can be used to determine the size of each unknown fragment using interpolation
• the fragments can also be removed and purified for further use
• gel electrophoresis can also be used to separate polypepetide fragments from protein molecules

 ·         for an animated view of gel electrophoresis, click on http://allserv.rug.ac.be/~avierstr/principles/electroani.html, and for an animated tutorial on gel electrophoresis, click on http://www.dnaftb.org/dnaftb/24/animation/index.html

 Homework:  11-14, p. 284

 Plasmids

 ·         cutting out sections of DNA from an existing genome, separating them, and then annealing them into another DNA molecule is a common practice in molecular biology

·         sometimes, gene fragments are excised for the purpose of inserting them into other genomes so that the genes can be expressed as useful protein

·         bacteria often provide the necessary machinery to carry out such an event

·         for example, the gene for insulin has been isolated and can now be expressed by bacterial cells

·         basically, the bacteria incorporates this gene within its genome, and expresses it by manufacturing insulin

·         this is where most of the insulin that diabetics use comes from

·         before this technology, diabetics used insulin made from animals, which caused allergic reactions

·         the expression of foreign genes is done through the insertion of DNA fragments into plasmids – small, circular, double-stranded DNA molecules that exist within bacterial strains

·         they are independent of the chromosome of the bacterial cell and range in size from 1000 to 200 000 base pairs

·         using enzymes and ribosomes that the bacterial cell houses, DNA contained in plasmids can be replicated and expressed

·         Figure 9, p. 285, shows the coexistence of bacterial DNA and plasmids – an mutualistic endosymbiotic relationship

·         plasmids consist of genes that express proteins able to confer antibiotic resistance (Figure 10, p. 285)

·         they also contain genes for resistance of toxic heavy metals, such as mercury, lead or cadmium, and contain genes that are able to break down herbicides, certain industrial chemicals, or the components of petroleum

·         Figure 10, p. 285 illustrates the genetically engineered plasmid pBR322 – as in most plasmids, it contains genes that confer antibiotic resistance (tet^r, and amp^r), a region that directs the plasmid’s replication called an origin of replication (ori), and recognition sites for restriction enzymes

·         plasmids also possess a characteristic copy number – the higher the number, the higher the number of individual plasmids in a host bacterial cell

·         the more plasmids in a cell, the more proteins are produced since more genes exist to make them

·         foreign genes are spliced into plasmids by using restriction endonucleases

·         artificial plasmids have been created that possess a unique region that can be cut by many restriction enzymes

·         the recognition sites of many restriction enzymes have been positioned very close together in this one area and are not found anywhere else on the plasmid’s DNA sequence – the site is called the multiple-cloning site

·         the recognition site exists in only one area of the plasmid which means that the DNA can only be cut at one location

·         one cut makes the plasmid linear

·         if the foreign gene has been excised using the same restriction enzyme, it will possess the same complementary ends as the linearized plasmid – this means that when they are mixed together, the linear sticky ends will anneal, making the foreign gene a permanent addition to the plasmid once the phosphodiester bonds are created (see Figure 11, p. 286)

·         DNA ligase does the phosphodiester linking of the sticky ends

·         the plasmid is now considered recombinant DNA – a combination of the original plasmid DNA and the foreign DNA

·         once this plasmid is introduced into a host bacterial cell, it will replicate to form many copies of itself, meaning it has been successfully cloned

·         many copies of the original gene can be produced this way, as well as the expression of the gene – a particular phenotype caused by a specific protein

Homework:  p. 287, 16-18 

 Transformation

·         the introduction of DNA from another source is known as transformation

·         the bacteria that has accepted a foreign plasmid is referred to as being transformed

·         this means that plasmids are used as vectors that carry desired genes into a host cell

·         bacteria that readily take up foreign DNA are called component cells

·         with the use of calcium chloride bacteria can be induced to accept plasmids into their cytoplasm and incorporate them into their cellular processes

·         calcium chloride ionizes into Ca²⁺ and Cl^- ions

·         bacterial cells are suspended in a solution of CaCl₂ at 0°C

·         the membrane of bacteria contain exposed negatively charged phosphates

·         the positive calcium ions stabilize the negative membrane phosphates, and the cold temperatures freeze the membrane, making it more rigid in structure

·         once the bacterial cell membrane has been both chemically and physically stabilized, the plasmid DNA is introduced to the solution

·         calcium ions also stabilize the plasmid DNA’s phosphates

·         Figure 12, p. 287 illustrates the effects of calcium chloride on the bacterial membrane and the plasmid

·         at this point, the entire solution is heat shocked to 42°C for approximately 90 seconds

·         this means that the outside of the cell is higher in temperature than the inside, creating a draft, which sweeps the plasmids into the bacteria cell through pores in the membrane

·         the bacteria are then incubated in a nutrient media suspension at a temperature of 37°C

·         selective plating isolates transformed bacteria from non-transformed bacteria

·         the vectors used for cloning carry an antibiotic-resistant gene – therefore, if the bacteria was successfully transformed, the bacteria would be able to grow on media that contain the antibiotic, and the non-transformed bacteria would not survive the antibiotic

·         Figure 13, p. 288 illustrates the selective plating technique

·         the existence of the foreign gene must also be tested to see if transformation was successful

·         the plasmid DNA is removed from the bacteria and is subjected to restriction enzyme digestion to release the cloned fragment from the vector

·         if the DNA shows the expected pattern on gel electrophoresis, then the colony of bacteria was successfully transformed and carries a recombinant DNA plasmid

·         other techniques, such as the use of electroporators that electrically shock the membranes, creating gaps where foreign DNA can enter, and “gene guns” that “shoot” DNA through the cell wall and membrane are used to transform bacteria as well

·         Table 2, p. 289, lists the three “tools” that are used in molecular biology, their use, and an example of each tool

 Homework:  p. 289, 19-21

 Major Steps in the Cloning of DNA (Figure 14, p. 291)

1. DNA fragments are generated

• Appropriate restriction endonucleases need to be used to ensure that the gene fragment in question is excised completely from the source of DNA – more than one restriction endonuclease may be used at one time

2. Recombinant DNA molecule is made

·         The target gene fragment is ligated to a DNA vector (i.e. a plasmid), making a recombinant DNA molecule

·         The DNA recombinant molecule replicates itself autonomously

·         The following is an example of a DNA vector with desired genetic information and an antibiotic resistant gene:

http://www.mun.ca/biology/scarr/Fg15_07.gif

3. Host cell receives recombinant DNA

·         Manipulation of bacterial cell membrane to take up the recombinant DNA by using elctroporators, gene guns, or classical transformation protocols, like calcium chloride: 

http://www.mun.ca/biology/scarr/Fig15-14_DNA_transformation.gif

·         The incorporation of the recombinant DNA into the bacteria cell so it actually becomes part of the bacteria’s cellular processes

        4.     The selection of the successfully transformed cells

·         Isolation of successfully transformed bacterial cells with the recombinant DNA using a marker, such as antibiotic resistance genes

·         If colonies grow, despite the existence of such antibiotics, then the recombinant DNA vector was successfully transformed

·         The following diagram illustrates the concept of colony plating:  http://www.mun.ca/biology/scarr/blue_and_white_colonies.gif and http://www.mun.ca/biology/scarr/Fg15_15smc.gif

·         The surviving colonies are isolated and are grown in culture to produce multiple copies of the incorporated recombinant DNA:  http://www.mun.ca/biology/scarr/Fig14-06_bacterial_amplification.gif

·         the following diagram summarizes the entire process:  http://www.mun.ca/biology/scarr/Fg15_14.gif

Homework:  p. 291, 1-17

·        for an excellent animation on recombinant DNA technology, click on http://present.smith.udel.edu/biotech/rDNA.html