Mary K. Campbell Shawn O. Farrell �������������������������� Chapter Thirteen Nucleic Acid Biotechnology Techniques Paul D. Adams • University of Arkansas 1
Purification and Detection of Nucleic Acids • Gel electrophoresis is a common technique used to separate used to separate nucleic acids. • Based on motion of charged particles in an electric field 2
Purification and Detection (Cont’d) Radioactive labeling of sample used to detect products • Label or tag allows visualization • DNA undergo reaction that incorporate radioactive isotope into the DNA • Autoradiography used to visualize image that has been exposed to • oligonucleotides that have been radiolabeled Fluorescence also used. Ethidium Bromide…can slip between DNA • bases, and it has different fluorescence characteristics as opposed to when it is free in solution EtBr is used as stain for DNA on gels. EtBr is dangerous ( a • carcinogen)…new fluorescent dyes have been developed (SyBr Green and Gold) 3
Restriction Endonucleases • Nucleases- catalyze the hydrolysis of the phosphodiester backbone of nucleic acids - Endonuclease : cleavage in the middle of the chain - Exonuclease : cleavage from the ends of the molecule • Restriction Endonucleases- Have a crucial role in development of recombinant DNA technology • Bacteriophages , viruses that infect bacteria, were being studied when restriction enzymes were discovered 4
Methylation of DNA 5
Restriction Endonucleases (Cont’d) • Restriction endonuclease (RE) hydrolyzes only a specific bond of a specific sequence in DNA • Sequences recognized by RE read the same from left to right as from right to left, known as palindrome • Two As and 2 Ts between breaks in DNA strand which leave sticky ends • Sticky ends are joined by by hydrogen bonding between complementary bases. • Ligases reseal ends 6
Restriction Endonucleases and Their Cleavage Sites 7
Action of DNA Ligases 8
Cloning • Recombinant DNA- DNA molecules that contain covalently linked segments derived from 2 or more DNA sources • Sticky Ends can be used to construct Recombinant DNA • DNA Ligase- seals nicks in the covalent structure • Plasmid- small circular DNA that is not part of the main circular DNA chromosome of the bacterium. circular DNA chromosome of the bacterium. • Cloning- The process of making identical copies of DNA 9
Production of Recombinant DNA 10
Plamids • How do we know which bacteria takes up the desired plasmid? • Selection- Each plasmid chosen for cloning has a selectable marker that indicates that the growing bacteria colonies contain the plasmid of interest 11
Plasmid pBR322 • One of the first plasmids used for cloning 12
Plasmids (Cont’d) • As the technology to design plasmids improved, regions were created that had many different restriction sites in a small place restriction sites in a small place • This region is known as a multiple cloning site (MCS), or polylinker 13
Cloning with pUC Plasmids 14
Blue/White Screening • Basis for selection • pUC plasmids contain lacZ gene • pUC plasmids contain lacZ gene • lacZ gene codes for the α -subunit of β - galactosidase, which cleaves disaccharides • This procedure helps with selection 15
Clone Selection with Blue/White Screening IPTG – isopropylthiogalactosie – lactose analogue that also binds to lac repressor X-gal - reagent that produces blue colour when converted by β -galactosidase 16
Cloning Summary • Cloning refers to creating identical populations • DNA can be combined by using restriction enzymes • The target DNA sequence is carried in some type of • The target DNA sequence is carried in some type of vector • The target DNA sequence is inserted into host organism • Organisms that carry the target DNA are identified through a process called selection through a process called selection 17
Genetic Engineering • When an organism is intentionally altered at the molecular level to exhibit different traits, it has been genetically engineered genetically engineered • One focus of genetic engineering has been gene therapy , where cells of specific tissues in a living person are altered in a way that alleviates the affects of a disease • DNA recombination can occur in nature DNA recombination can occur in nature • The reproductive power of bacteria can be used to express large quantities of a mammalian protein of interest, however, process can be complicated 18
Cloning Vectors • Plasmid vectors pBR322 and pUC are cloning vectors • Vectors are used to insert foreign DNA and amplify it • Vectors are used to insert foreign DNA and amplify it • If we want to produce produce protein from the foreign DNA, vectors are not good 19
Expression Vectors • Have many attributes as cloning vector: • The origin of replication • A multiple cloning site • A multiple cloning site • At least one selectable marker • Must be able to be transcribed by the genetic machinery of the bacteria where it is transformed • Must have a transcription termination sequence 20
DNA Libraries • Can we take all the DNA of an organism and clone it in chunks and clone it in chunks of reasonable size • The result of this is a DNA library • Several steps involved in construction of the in construction of the library 21
Finding an Individual Clone in a DNA Library • After the library has been constructed, the next challenge is to find a challenge is to find a single desired clone out of hundreds of thousands, or millions Technique used to • select depends on separating and annealing complementary strands • Known as Genomic Library Screening 22
Finding an Individual Clone in a DNA Library (Cont’d) • RNA libraries not constructed in the same way • RNA of interest is used as template for the synthesis of complementary DNA (cDNA) • Reaction catalyzed by reverse transcriptase • cDNA is incorporated into vector, then process is identical to the production of genomic DNA library 23
Summary • A DNA library is a collection of clones of an entire genome • The genome is digested with restriction enzymes • The genome is digested with restriction enzymes and the pieces are cloned into vectors, and transformed into cell lines • Specific radioactive probes to a sequence of interest are reacted to filters that have copies of the bacterial colonies in the library colonies in the library • A cDNA library is constructed by using reverse transcriptase to make DNA from the mRNA in a cell. This cDNA is then used to construct a library similar to a genomic DNA library 24
The Polymerase Chain Reaction • It is possible to increase the amount of a given DNA many times over without cloning the DNA • This method of amplification is known as the Polymerase Chain Reaction (PCR) • Any chosen DNA can be amplified, and it does not need to be separated from the rest of the DNA in a need to be separated from the rest of the DNA in a sample before the procedure is applied 25
The Polymerase Chain Reaction (Cont’d) 26
DNA Fingerprinting • DNA samples can be studied and compared by DNA fingerprinting • DNA is digested with restriction enzymes and • DNA is digested with restriction enzymes and then run on an agarose gel • When soaked in ethidium bromide, the DNA fragments can be seen directly under UV light • If greater sensitivity needed or if number of fragments would be too great to distinguish the fragments would be too great to distinguish the bands, technique can be modified to show only selected DNA sequences • This begins with Southern blotting 27
The Southern Blot 28
Restriction-Fragment Length Polymorphisms • In organisms with two sets of chromosomes, a given gene on one chromosome may differ slightly from the corresponding gene on the paired chromosome • These are known as alleles • Organisms are homozygous when they have the same paired chromosomes • Organisms are heterozygous when they have different paired chromosomes • Restriction fragments of different sizes are obtained by treatment with endonuclease. They are Restriction- Fragment Length Polymorphisms (RFLPs) 29
The Basis for Restriction-Fragment Length Polymorphism 30
Summary • A DNA fingerprint is created by digesting DNA with restriction enzymes, separating the pieces on a gel, and visualizing some of the pieces by using labeled and visualizing some of the pieces by using labeled probes • Differences in DNA patterns between different individuals are based on different base sequences of their DNA their DNA 31
DNA Sequencing • The nature and order of monomer units determine the properties of the whole molecule • The method devised by Sanger and Coulson for • The method devised by Sanger and Coulson for determining the base sequences of nucleic acids depends on selective interruption of oligonucleotide synthesis • A single-stranded DNA fragment whose sequence is to be determined is used as a template to be determined is used as a template • The synthesis is interrupted at every possible site in the population of molecules depending on the presence of ddNTPs 32
Recommend
More recommend
