DNA Technology: Nucleic Acid Hybridization
o When heated or immersed in high concentration salt solution or high pH solution, the hydrogen bonds connecting the two strands in a double stranded DNA molecule are disrupted, and the strands separate; the DNA molecule is said to be denatured or melted.
o Denatured DNA is less viscous (less syrupy), denser, and more able to absorb UV light.
o Separated strands will spontaneously associate with their original partner or any complementary nucleotide sequence.
o Thus, the following double stranded combinations can be formed through nucleic acid hybridization.
o DNA-DNA, DNA-RNA, RNA-RNA
o One method bacteria use to defend themselves from viruses is to cut the viral DNA into fragments with restriction enzymes.
o The bacteria protect their own DNA from these enzymes by methylation.
o Methylation is usually, but not always, associated with inactivated genes.
o Restriction enzymes (also called restriction endonucleases) digest/cut nucleic acid only at certain nucleotide sequences along the chain.
o Such a sequence is called a restriction sire or recognition sequence.
o Typically, a restriction site will be a palindromic sequence four to six nucleotides long.
o Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single stranded ends.
o These ends can reconnect through hybridization and are termed sticky ends.
o Sticky ends are produced by cutting the DNA in a staggered manner within the recognition site producing single-stranded DNA ends.
o It can also be cut so that it has blunt ends.
o Two DNA fragments cleaved by the SAME endonuclease can be joined together regardless of the origin of the DNA.
o Such DNA is called recombinant DNA; it has been artificially recombined.
o A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.
o In many cases, it is circular and double-stranded.
o Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms.
o Plasmids used in genetic engineering are called vectors.
o Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.
o The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site, which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.
o Next, the plasmids are inserted into bacteria by a process called transformation.
o Then, the bacteria are exposed to the particular antibiotics.
o Only bacteria which take up copies of the plasmid survive , since the plasmid makes them resistant.
o Eukaryotic DNA contains introns.
o Since bacteria have no mechanism for removing introns, it is useful to clone DNA with no introns.
o In order to do this, the mRNA produced by the DNA is reverse transcribed using reverse transcriptase.
o The DNA product is called complementary DNA (cDNA).
o Adding DNA polymerase to cDNA produces a double strand of the desired DNA fragment (an entire DNA double helix free of introns).
Polymerase Chain Reaction (PCR)
o The polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence.
o The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.
o Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase.
o Almost all PCR applications employ a heat-stable DNA polymerase, are key components to enable selective and repeated amplification.
o As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.
o Starting with a single fragment, 20 cycles produces 2^20 copies
o Southern blotting is a technique used to identify target fragments of known DNA sequence in a large population of DNA.
o It is separated according to size by gel electrophoresis.
o The anode for Southern blotting is positive!.
o The cathode for Southern blotting is negative.
o Northern blotting is just like Southern blotting but it identifies RNA fragments.
Restriction Fragment Length Polymorphism (RFLP)
o Restriction fragment length polymorphism (RFLP) analysis identifies individuals as opposed to individual specific genes.
o The DNA of different individuals possesses different restriction sites and varying distances between restrictions sites.
o After fragmenting the DNA sample with endonucleases, southern blotting is used and produces bands distinct to the individual.
o The genome of one human differs from the next at about one nucleotide in every 1000.
o These differences have been called single nucleotide polymorphisms (SNPs)
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