2 Separation techniques of Nucleic Acids Gel electrophoresis -used to separate nucleic acids based on charge and size.Proteins – SDS PAGEDone in an electric field
3 Detection of Nucleic Acids Radioactive labeling of sample used to detect productsLabel or tag allows visualizationDNA undergoes reaction that incorporates radioactive isotope into the DNAAutoradiography used to visualize image that has been exposed to radiolabeled oligonucleotidesIsotope – p32 and S35. After exposure the gel is placed in contact with X-ray film.
4 Detection of Nucleic Acids FluorescenceEthidium bromide intercalates between basesUnder UV light glows orange
5 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 moleculeRestriction Endonucleases - Have a crucial role in development of recombinant DNA technologyBacteriophages - viruses that infect bacteria- Led to discovery of restriction enzymesNucleases are specific to DNA and some can be specific to RNA. One group of nucleases is RE.
7 Restriction Endonucleases Restriction endonucleases (RE) hydrolyzes only a specific bond of a specific sequence in DNASequences recognized by RE read the same from left to right as from right to left, known as palindromeSticky and Blunt ends
8 Resealing by DNA ligase Sticky ends are joined by hydrogen bonding between complementary bases.Ligases reseal ends
9 Recombinant DNA Technology Recombinant/Chimeric DNA - DNA molecules that contain covalently linked segments derived from 2 or more DNA sourcesSticky Ends can be used to construct Recombinant DNADNA Ligase- seals nicks in the covalent structure
14 pBR322 One of the first plasmids used for cloning – E.coli Foreign DNA must be inserted at unique restriction sitesConfers resistance to two antibiotics – Tetracycline and ampicillinOrigin of replication and replicated independently of the chromosomal DNA.
15 PlasmidsAs the technology to design plasmids improved, regions were created that had many different restriction sites in a small placeThis region is known as a multiple cloning site (MCS) or polylinkerpUC – universal cloning plasmid
16 Selection 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 bacterial colonies contain the plasmid of interest
17 Clone Selection with Blue/White Screening Basis for selectionpUC plasmids contain lacZ genelacZ gene codes for the -subunit of -galactosidase, which cleaves disaccharidesThis procedure helps with selection
18 Cloning Summary Cloning refers to creating identical populations DNA can be combined by using restriction enzymes + LigasesThe target DNA sequence is carried in some type of vector/plasmidThe target plasmid is inserted into host organismOrganisms that carry the target DNA are identified through a process called selection
19 Genetic EngineeringWhen an organism is intentionally altered at the molecular level to exhibit different traits - genetically engineeredOne 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
20 Protein Expression Vectors Plasmid vectors pBR322 and pUC are cloning vectorsVectors are used to insert foreign DNA and amplify itIf we want to produce protein from the foreign DNA - Expression vectors
21 What is an Expression Vector? Have many attributes as cloning vector:- The origin of replicationA multiple cloning siteAt least one selectable markerMust have a transcription termination sequence
22 What is an Expression Vector? Must be able to be transcribed by the genetic machinery of the bacteria where it is transformedMust have a transcription initiation and termination sequenceRibosomal binding site-translation
24 DNA librariesAll the DNA of an organism - clone it in chunks of reasonable sizeThe result of this is a DNA librarySeveral steps involved in construction of the library
25 How do we find the piece of DNA we want in a library? Genomic Library ScreeningA nitrocellulose disc is put on the dish and removedDisc treated with denaturing agent to unwind DNADNA is permanently fixed to disc by treatment with heat or UV light
26 How do we find the piece of DNA we want in a library? Expose DNA on disc to a solution that contains single stranded complementary DNA or RNA (radioactive probing)Wash the discIdentify the colonies
27 Making cDNA libraryRNA of interest is used as template for the synthesis of complementary DNA (cDNA)Reaction catalyzed by reverse transcriptasecDNA is incorporated into vectorcDNA library construction is identical to genomic DNA libraryRNA libraries not constructed in the same way
28 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 vectorsA 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
29 Polymerase Chain Reaction It is possible to increase the amount of a given DNA many times over without cloning the DNAAny chosen DNA can be amplified, and it does not need to be separated from the rest of the DNA in a sample
30 DNA fingerprintingDNA samples can be studied and compared by DNA fingerprintingDNA is digested with restriction enzymes and then run on an agarose gelWhen soaked in ethidium bromide – can be seen directly under UV light
31 Southern blottingIf 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
32 SequencingDNA can be sequenced by using several techniques, the most common being the chain termination methodDideoxy nucleotides are used to terminate DNA synthesis. Multiple reactions are run with different dideoxy nucleotide in each reaction mixThe 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
34 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 nowThe proteome is a protein version of a genomeProteomics is the study of interactions among all the proteins in a cell
35 Open book take home quiz for 30 points 1. Name the two kinds of gels used in electrophoresis and what molecules do those separate. Explain the original charges of those molecules and in which direction do they move in an electric field. In other words explain the effect of charge and size on the biomolecules.2. What is methylation of DNA in Bacteria?3. Write names of any two enzymes and the name of the bacteria from which it has been extracted. Explain how these enzymes have been named or their naming procedures.4. Draw a recombinant DNA plasmid showing the sites of BamHI and HindIII along with same RE sites on your DNA of interest.5. Explain the whole procedure of PCR along with factors required to run a PCR reaction.6. Explain the procedure of RNA interference.7. Explain the Agrobacterium transformation done in plants.
36 This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB ). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.