Purification and Detection of Nucleic Acids

2. Purification and Detection of Nucleic Acids • Gel electrophoresis is a common technique used to separate nucleic acids. • Based on motion of charged particles in an electric field

3. 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

4. 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

5. Methylation of DNA

6. 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

7. Restriction Endonucleases and Their Cleavage Sites

8. Action of DNA Ligases

9. 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. • Cloning- The process of making identical copies of DNA

10. Production of Recombinant DNA

11. The Cloning of a Virus

12. 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

13. Plasmid pBR322 • One of the first plasmids used for cloning

14. Plasmids (Cont’d) • As the technology to design plasmids improved, regions were created that had many different restriction sites in a small place • This region is known as a multiple cloning site (MCS), or polylinker

15. Blue/White Screening • Basis for selection • pUC plasmids contain lacZ gene • lacZ gene codes for the -subunit of -galactosidase, which cleaves disaccharides • This procedure helps with selection

16. Clone Selection with Blue/White Screening

17. 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 vector • The target DNA sequence is inserted into host organism • Organisms that carry the target DNA are identified through a process called selection

18. Genetic Engineering • When an organism is intentionally altered at the molecular level to exhibit different traits, it has been 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 • The reproductive power of bacteria can be used to express large quantities of a mammalian protein of interest, however, process can be complicated

19. Genetic Engineering (Cont’d) • Human proteins can be made by bacteria, but process is not straight forward. e.g. human insulin • An intron is a DNA sequence that codes for RNA that is eventually deleted in the processing of the mRNA that directs the synthesis of the protein • Only the RNA transcribed from exons appear in the mature RNA

20. Protein Expression Vectors • Plasmid vectors pBR322 and pUC are cloning vectors • 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 • Instead, expression vectors are used

21. What is an Expression Vector? • Have many attributes as cloning vector: • The origin of replication • 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

22. DNA Libraries • Can we take all the DNA of an organism and clone it in chunks of reasonable size • The result of this is a DNA library • Several steps involved in construction of the library

23. Finding an Individual Clone in a DNA Library • After the library has been constructed, the next 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

24. 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

25. Summary • A DNA library is a collection of clones of an entire genome • 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 • 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

26. 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 sample before the procedure is applied

27. The Polymerase Chain Reaction (Cont’d)

28. DNA Fingerprinting • DNA samples can be studied and compared by DNA fingerprinting • 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 bands, technique can be modified to show only selected DNA sequences • This begins with Southern blotting

29. The Southern Blot

30. 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)

31. The Basis for Restriction-Fragment Length Polymorphism

32. 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 probes • Differences in DNA patterns between different individuals are based on different base sequences of their DNA

33. DNA Sequencing • The nature and order of monomer units determine the properties of the whole molecule • 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 • The synthesis is interrupted at every possible site in the population of molecules depending on the presence of ddNTPs

34. DNA Sequencing (Cont’d) • The incorporation of the ddNTP into the growing chain causes termination at the point of incorporation • The DNA to be sequenced is mixed with a short oligonucleotide that serves as a primer for synthesis of the complementary strand • Gel electrophoresis is performed on each reaction mixture, and a band corresponding to each position of the chain termination appears • The sequence of the newly formed strand, complementary to the template DNA, can then be read from the sequencing gel

35. The Sanger-Coulon Method for Sequencing DNA

36. Summary • DNA can be sequenced by using several techniques, the most common being the chain termination method • Dideoxy nucleotides are used to terminate DNA synthesis. Multiple reactions are run with different dideoxy nucleotide in each reaction mix • The reactions produce a series of DNA fragments of different length that can be run on a gel and the sequence determined by tracking the different length fragments in the lanes with the four different dideoxy nucleotides

37. Genomics and Proteomics • Knowing the full DNA sequence of the human genome allows for the investigation for the causes of disease in a way that has not been possible until now • The proteome is a protein version of a genome • Proteomics is the study of interactions among all the proteins in a cell

38. Microarrays

39. Summary • As more DNA sequences become available, it becomes possible to compare these sequences • Important medical applications are emerging, and new methods are making it possible to analyze large quantities of data • The proteome is the protein version of the genome. It refers to all of the proteins being expressed in a cell

40. Summary • DNA or protein microchips is a powerful technique being used presently, as thousands of samples of DNA or proteins can be applied and then checked for binding of biological samples • The binding can be visualized by using fluorescently labeled molecules and scanning the chip with a computer (Figure 13.30). The pattern of fluorescent labels then indicates which mRNA or proteins are being expressed in the samples