History of Molecular Genetics and Biotechnology Help (page 3)

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By — McGraw-Hill Professional
Updated on Apr 25, 2014

Fluorescent Labels

Fluorescent molecules contain a fluorophore that absorbs and emits light at particular wavelengths characteristic to the specific fluorophore. This emitted light can be detected and recorded by a number of different instruments (microscopes, scanners, spectrophotometers, etc). Fluorescent molecules (probes) can be attached to a variety of biological molecules in order to detect and study them. DNA, proteins, and lipids, as well as specific molecules, such as actin and tubulin, can be labeled with a fluorescent molecule. It is possible to use two or more fluorescent probes in one experiment if their emitted light occurs at different wavelengths. For example, different molecules within a cell can each be labeled with a different fluorescent molecule and the activities and amounts of each molecule can be detected and compared at the same time. During DNA sequencing reactions, each nucleotide is labeled with a different fluorescent molecule, allowing strands terminating with a different nucleotide to be differentiated yet detected simultaneously (see section on DNA Sequencing later in this chapter).

Nucleic Acid Enzymology

Nucleases are enzymes that hydrolyze, or break, the phosphodiester bonds that hold the nucleotides together. Those that remove terminal nucleotides one at a time are called exonucleases; those that break the sugar-phosphate backbone at nonterminal sites are called endonucleases.Adeoxyribonuclease (DNase) degrades DNA molecules; a ribonuclease (RNase) degrades RNA molecules. Some endonucleases act nonspecifically, cleaving the phosphodiester bonds at different unspecified nucleotide sequences. Others, such as the restriction endonucleases, break the bonds only at specific DNA sequences, called recognition sites. There are three classes of restriction endonucleases: Types I, II and III. Types I and III do not have qualities useful for recombinant DNA technology (i.e., they cut DNA at random sites). Type II enzymes recognize and bind to a specific double-stranded DNA sequence. Once bound, they cleave the phosphodiester backbone of each strand at or near (within 20 bp) the site. The recognition sites of these enzymes are symmetrical and commonly consist of 4–8 bp that are either continuous (e.g.,GAATTC) or interrupted (e.g., CANTG, where N is any base). The symmetry occurs around a midpoint, or axis of symmetry, formed on opposite DNA strands by inverted base sequences called palindromes. These enzymes occur naturally in bacteria, and hundreds of enzymes have been isolated and characterized and are available for use by genetic engineers. Thus, Type II enzymes are the most commonly used in recombinant DNA technology today.

Restriction enzymes play a role in defending bacteria against invasion by foreign nucleic acids, such as viruses. The host bacterial cell restriction enzyme systems can recognize the invading DNA as foreign and destroy it (see Example 11.1). Host DNA may be protected by specific base-pair modifications carried out by host-specific methylase enzymes. Thus, the restriction enzyme works in conjunction with the modifying enzyme and together the system is referred to a restriction-modification system. Foreign DNA is recognized as such because its restriction sites are unmodified. Restriction endonucleases are named after the bacterial species or strain from which they were derived. For example, an enzyme from Providentia stuartii, will have the name Pst derived from the first letter of the genus (P) and the first two letters of the specific epithet (st). This name is italicized to honor the scientific name of the bacterium and can be followed by roman numerals to indicate that it is one of several enzymes isolated from that particular bacterial strain, e.g. PstI, PstII (the numerals are not italicized). Occasionally, a letter derived from the specific bacterial strain follows the name. For example, the enzyme EcoRI was derived from E. coli strain RY13 and Hind III was derived from Haemophilus influenzae strain Rd. If this is the case neither the strain letter nor the numerals are italicized.

EXAMPLE 12.2 The EcoRI restriction enzyme cuts bonds on the upper and lower strands within the palindromic DNA sequence at the arrows shown below.


Notice that the 5' to 3' nucleotide sequence within the palindrome is the same starting from the 5' (pronounced five prime) ends on both strands of the DNA (the palindrome). EcoRI cuts the DNA molecule in a staggered fashion, leaving "overhang,'' "cohesive'' or "sticky'' ends. In the above example, the overhangs are called 5' overhangs because the unbonded nucleotides have a 5' end. Other restriction enzymes can leave a 3' overhanging end. Another restriction endonuclease (HaeIII), derived from the bacterium Haemophilus aegypticus, snips DNA as shown below. Note that this enzyme cuts both strands at opposite bonds, leaving "blunt" ends.


Restriction enzyme maps for any given DNA segment (linear or circular) can be constructed. Such maps show the location of various restriction endonuclease recognition sites on the DNA fragment. The sizes of the various restriction fragments can be expressed by molecular weight, but more commonly they are given in terms of number of base pairs (bp) or thousands of base pairs (kilobases, kb).

EXAMPLE 12.3 A linear 10,000-bp fragment of DNA has an unknown number of EcoRI restriction enzyme sites. You perform a restriction digestion of this fragment with EcoRI and obtain the following result: three fragments of sizes 5, 3, and 2 kb. These results suggest that there are two EcoRI sites present in this molecule. One possible arrangement of sites (arrows) is shown below. There are two other possible arrangements (not shown).


If this molecule were circular, as are bacterial plasmids commonly used in recombinant DNA technology, only two fragments (3 and 7 kb) would be obtained. The 7 kb fragment is a result of the joining of the 5 and 2 kb segments (see diagram below).


Many other enzymes that are involved in the replication, recombination, repair, modification, transcription, and translation of nucleic acids are utilized in recombinant DNA technology. In particular, DNA synthesis enzymes, known as DNA polymerases, have become very useful for synthesizing copies of DNA molecules in vitro (in a test tube) in a process called the polymerase chain reaction, or PCR (see later section).

Practice problems for these concepts can be found at:

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