1. Chp 8 DETECTION OF GENES AND GENE PRODUCTS Huseyin Tombuloglu PhD. GBE310, Spring 2015

2. Molecular geneticists usually want to study particular genes within the chromosomes of living species –This presents a problem, because chromosomal DNA contains thousands of different genes –The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes Scientists have also developed techniques to identify gene products –RNA that is transcribed from a particular gene –Protein that is encoded in an mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DETECTION OF GENES AND GENE PRODUCTS 18-44

3. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A DNA library is a collection of thousands of different cloned fragments of DNA made by cutting up the genome of an organism When the starting material is chromosomal DNA, the library is called a genomic library A cDNA library contains hybrid vectors with cDNA inserts Should represent the genes expressed in the cells the RNA was isolated from The construction of a DNA library is shown in Figure 18.7 DNA Libraries 18-45

4. 18-46 Figure 18.7

5. 18-47 Figure 18.7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6. In most cloning experiments, the ultimate goal is to clone a specific gene For example, suppose that a geneticist wishes to clone the rat  -globin gene Only a small percentage of the hybrid vectors in a DNA library would actually contain the gene Therefore, geneticists must have a way to distinguish those rare colonies from all the others This can be accomplished by using a DNA probe in a procedure called colony hybridization Refer to Figure 18.8 18-48

7. 18-49 Figure 18.8

8. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display But how does one obtain the probe? If the gene of interest has been already cloned, a piece of it can be used as the probe If not, one strategy is to use a probe that likely has a sequence similar to the gene of interest For example, use the rat  -globin gene to probe for the  -globin gene from another rodent What if a scientist is looking for a novel type of gene that no one else has ever cloned from any species? If the protein of interest has been previously isolated, amino acid sequences are obtained from it The researcher can use these amino sequences to design short DNA probes that can bind to the protein’s coding sequence 18-50

9. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Southern blotting can detect the presence of a particular gene sequence within a mixture of many It was developed by E. M. Southern in 1975 Southern blotting has several uses 1. It can determine copy number of a gene in a genome 2. It can detect small gene deletions that cannot be detected by light microscopy 3. It can identify gene families 4. It can identify homologous genes among different species Southern Blotting 18-51

10. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene, has been cloned This cloned DNA is labeled (e.g., radiolabeled) and used as a probe The probe will be able to detect the gene of interest within a mixture of many DNA fragments The technique of Southern Blotting is shown in Figure 18.9 18-52

11. 18-53 An alternative type of transfer uses a vaccuum Figure 18.9

14. 18-54 Figure 18.9 a) The steps in Southern blotting A common labeling method is the use of the radioisotope 32 P Conditions of high temperature or high salt concentrations Probe DNA and chromosomal fragment must be nearly identical to hybridize Temperature and/or ionic strength are lower Probe DNA and chromosomal fragment must be similar but not necessarily identical to hybridize Gene of interest is found only in single copy in the genome Gene is member of a gene family composed of three distinct members Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15. Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules It was not named after anyone called Northern! Originally known as ‘Reverse-Southern’ which became Northern. Northern blotting has several uses 1. It can determine if a specific gene is transcribed in a particular cell type Nerve vs. muscle cells 2. It can determine if a specific gene is transcribed at a particular stage of development Fetal vs. adult cells 3. It can reveal if a pre-mRNA is alternatively spliced Northern Blotting 18-55

16. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Northern blotting is rather similar to Southern blotting It is carried out in the following manner RNA is extracted from the cell(s) and purified It is separated by gel electrophoresis It is then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a radioactive probe The filters are then exposed to an X-ray film RNAs that are complementary to the radiolabeled probe are detected as dark bands on the X-ray film Figure 18.10 shows the results of a Northern blot for mRNA encoding a protein called tropomyosin 18-56

17. 18-57 Figure 18.10 Smooth and striated muscles produce a larger amount of tropomyosin mRNA than do brain cells This is expected because tropomyosin plays a role in muscle contraction The three mRNAs have different molecular weights This indicates that the pre-mRNA is alternatively spliced Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18. Western blotting is used to identify a specific protein within a mixture of many protein molecules Again, it was not named after anyone called Western! Western blotting has several uses 1. It can determine if a specific protein is made in a particular cell type Red blood cells vs. brain cells 2. It can determine if a specific protein is made at a particular stage of development Fetal vs. adult cells Western Blotting 18-58

19. Western blotting is carried out as follows: Proteins are extracted from the cell(s) and purified They are then separated by SDS-PAGE They are first dissolved in the detergent sodium dodecyl sulfate This denatures proteins and coats them with negative charges The negatively charged proteins are then separated by polyacrylamide gel electrophoresis They are then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a primary antibody (recognizes the protein of interest) A secondary antibody, which recognizes the constant region of the primary antibody, is then added The secondary antibody is also conjugated to alkaline phosphatase The colorless dye XP is added Alkaline phosphatase converts the dye to a black compound Thus proteins of interest are indicated by dark bands 18-59

20. 18-60 Figure 18.11 shows the results of a Western blot for the  -globin polypeptide This experiment indicates that  -globin is made in red blood cells but not in brain or intestinal cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21. Researchers often want to study the binding of proteins to specific sites on a DNA molecule For example, the binding to DNA of transcription factors To study protein-DNA interactions, the following two methods are used 1. Gel retardation assay Also termed band shift assay 2. DNA footprinting Techniques that Detect the Binding of Proteins to DNA 18-61

22. 18-62 Figure 18.12 The technical basis for a gel retardation assay is this: The binding of a protein to a fragment of DNA retards its rate of movement through a gel Gel retardation assays must be performed under nondenaturing conditions Buffer and gel should not cause the unfolding of the proteins nor the separation of the double helix Higher mass and therefore slow migration Lower mass and therefore fast migration Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

23. DNA footprinting was described originally by David Galas and Albert Schmitz in 1978 They identified a DNA site in the lac operon that is bound by the lac repressor This DNA site is, of course, the operator The technical basis for DNA footprinting is this: A segment of DNA that is bound by a protein will be protected from digestion by the enzyme DNase I Figure 18.13 shows a DNA footprinting experiment involving RNA polymerase holoenzyme 18-63

24. 18-64 Figure 18.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Did not ontain RNA pol holoenzyme

25. 18-65 Figure 18.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the absence of RNA pol holoenzyme, a continuous range of sizes occurs No bands in this range RNA pol holoenzyme is bound to this DNA region, and thus protects it from DNase I Thus RNA pol holoenzyme binds to an 80-nucleotide region (from -50 to +30)

26. Analyzing and altering DNA sequences is a powerful approach to understanding genetics –A technique called DNA sequencing enables researchers to determine the base sequence of DNA It is one of the most important tools for exploring genetics at the molecular level –Another technique known as site-directed mutagenesis allows scientists to change the sequence of DNA This too provides information regarding the function of genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18.3 ANALYSIS & ALTERATION OF DNA SEQUENCES 18-66

27. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During the 1970s two DNA sequencing methods were devised One method, developed by Alan Maxam and Walter Gilbert, involves the base-specific cleavage of DNA The other method, developed by Frederick Sanger, is known as dideoxy sequencing The dideoxy method has become the more popular and will therefore be discussed here DNA Sequencing 18-67

28. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The dideoxy method is based on our knowledge of DNA replication but uses a clever twist DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other (Refer to Fig. 11.10) Nucleotides missing that 3’–OH can be synthesized 18-68 Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow This is referred to as chain termination If ddATP is used, termination will always be at an A in the DNA Figure 18.14

29. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts This is accomplished using cloning or PCR techniques In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site In the experiment shown in Figure 18.15, the vector DNA is from a virus called M13 After cloning, the viral DNA is introduced into the host cell There it will produce single-stranded DNA as part of its life cycle If double-stranded DNA is used as the template, it must be denatured at the beginning of the experiment 18-69

30. 18-70 Figure 18.15 The newly-made DNA fragments can be separated according to their length by running them on an acrylamide gel They can then be visualized as bands when the gel is exposed to X-ray film Sequencing ladder

31. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An important innovation in the method of dideoxy sequencing is automated sequencing It uses a single tube containing all four dideoxyribonucleotides However, each type (ddA, ddT, ddG, and ddC) has a different- colored fluorescent label attached After incubation and polymerization, the sample is loaded into a single lane of a gel 18-71 Figure 18.16

32. The procedure is automated using a laser and fluorescent detector The fragments are separated by gel electrophoresis Indeed, the mixture of DNA fragments are electrophoresed off the end of the gel As each band comes off the bottom of the gel, the fluorescent dye is excited by the laser The fluorescence emission is recorded by the fluorescence detector The detector reads the level of fluorescence at four wavelengths 18-72 Figure 18.16

33. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Analysis of mutations can provide important information about normal genetic processes Therefore, researchers are constantly looking for mutant organisms Mutations can arise spontaneously, or be induced by mutagens Researchers have recently developed techniques to make mutations within cloned DNA Site-Directed Mutagenesis 18-73

34. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display One widely-used method is known as site-directed mutagenesis It allows the alteration of a DNA sequence in a specific way The site-directed mutant can then be introduced into a living organism This will allow the researchers to see how the mutation affects The expression of a gene The function of a protein The phenotype of an organism Mark Zoller and Michael Smith developed a protocol for the site-directed mutagenesis of DNA cloned in a viral vector Refer to Figure 18.17 18-74

35. 18-75 Figure 18.17 The vector is the M13 virus which produces a single-stranded DNA as part of its life cycle Depending on which base is replaced, the mutant or original sequence is produced Can be identified by DNA sequencing and used for further studies