Chap. 5 Molecular Genetic Techniques (Part B)

1. Chap. 5 Molecular Genetic Techniques (Part B) • Topics • DNA Cloning and Characterization (cont.) • Using Cloned DNA fragments to Study Gene Expression Goals Learn genetic and recombinant DNA methods for isolating genes and characterizing the functions of the proteins they encode. Use of RNA interference (RNAi) in analysis of planarian regeneration

2. Separation of DNA Fragments by Gel Electrophoresis DNA fragments must be separated and purified prior to many rDNA procedures. A convenient method for both separation and purification is gel electrophoresis. DNA molecules have a uniform charge to mass ratio. In an electric field they run towards the anode and are separated based on size (length) when electrophoresed through a gel sieving network made of polyacrylamide or agarose (Fig. 5.19a). DNA bands can be visualized by radiolabeling the DNA or by noncovalent binding of the fluorescent dye known as ethidium bromide (Fig. 5.19b). The region of the gel containing the band can be excised and the DNA fragment obtained by extraction with a buffer.

3. Polymerase Chain Reaction (Part 1) The PCR is a method for amplifying a DNA sequence region located between two primers (Fig. 5.20). Amplification is specific and highly sensitive, allowing a target sequence to be specifically amplified starting from a complex mixture of DNA. In Cycle 1, double-stranded DNA containing the target sequence is first denatured by heating to >90˚C, PCR primers are annealed by reducing the temperature to ~ 50-60˚C, and then the primers are elongated by a DNA polymerase. The process is repeated over many cycles (next slide).

4. Polymerase Chain Reaction (Part 2) In later cycles, most of the DNA synthesized corresponds exclusively to the sequence region between the primers. The yield of amplified DNA increases exponentially based on the cycle number, n (yield is proportional to 2n). A heat-resistant DNA polymerase, typically Taq polymerase from the Yellowstone Archaen organism, Thermus aquaticus, is used as the DNA polymerase to prevent its denaturation due to heating.

5. Cloning of PCR-amplified DNA PCR fragments can be readily cloning into a vector by incorporating restriction enzyme sites into the ends of the primers used in amplification (Fig. 5.21). The added sequences do not interfere with polymerization reactions.

6. DNA Sequencing (Part 1) The classical method for DNA sequencing is dideoxy chain-termination sequencing (Sanger sequencing). The basic approach involves 1) enzymatic synthesis of a set of specifically labeled (at each base) daughter strands from the molecule being sequenced that differ by one nucleotide in length, and 2) separation of the fragments by electrophoresis. The sequence then is read from the positions of consecutive fragments on the gel. Termination at each base is accomplished using dideoxyribonucleoside triphosphates (ddNTPs) (figure). These nucleotides are incorporated into a growing DNA chain, but block further elongation because they lack a 3'-hydroxyl group.

7. DNA Sequencing (Part 2) Chain-termination strand synthesis by a DNA polymerase is illustrated for the G reaction in the figure at left below. To prevent all chains from terminating at the first G position, ddGTP is added at ~1/100th the amount of dGTP. To achieve termination at each type of base, four separate reactions are run in parallel using the sequencing template (right, below). Each reaction is spiked with one of the four ddNTPs.

8. DNA Sequencing (Part 3) The Sanger method of DNA sequencing is being replaced by so-called next generation sequencing, which has a greater capacity for sequence generation. Next generation sequencing currently is the preferred method for sequencing of entire genomes. In the method, a large collection of DNA fragments generated from genomic DNA, for example, is prepared and attached to a solid support (Fig. 5.23). PCR is used to amplify each attached fragment into a cluster of about 1,000 molecules. As many as 3 x 109 discrete clusters are attached to the final support. Each cluster derives from a unique fragment in the original collection.

9. DNA Sequencing (Part 4) The DNA fragments in each cluster then are sequenced using fluorescently-tagged dNTPs, in which each species is tagged with a different color (Fig. 5.24, top). dNTPs are incorporated one at a time in each cycle of sequencing, and the color and identity of the dNTP is determined by fluorescent microscopy (Fig. 5.24, bottom). Up to 100 cycles of sequencing are performed. Thus, 3 x 1011 total bases of sequence information can be collected from the 3 x 109 clusters attached to the support. The sequences of all fragments are aligned to determine overlapping regions, and the overlaps are used to assemble the overall sequence of the starting genomic DNA, for example.

10. Southern Blotting Southern blotting is a sensitive method for the detection of a DNA sequence within a mixture (Fig. 5.26). The DNA first is cleaved with a restriction enzyme to produce fragments that can be separated by electrophoresis. After electrophoresis, DNA fragments are denatured with alkali and transferred to a nitrocellulose membrane by capillary action, creating a replica of the original gel. The membrane is incubated with a labeled probe which binds to and detects the fragment of interest.

11. Northern Blotting Northern blotting is a method for the detection of a specific mRNA in a mixture of RNAs. Like Southern blotting the method is highly specific and sensitive. RNA from a cell/tissue is extracted and separated by electrophoresis. As in Southern blotting the RNA is transferred to a nitrocellulose membrane and incubated with a labeled probe that is complementary to the RNA. As shown in Fig. 5.27, the method can be use to quantitate transcription of a mRNA under different cellular conditions.

12. DNA Microarrays and Transcriptome Analysis A complete analysis of all the mRNAs transcribed in an organism (transcriptome) can be performed by DNA microarray analysis (Fig. 5.29). In this method, mRNA is isolated and converted to cDNA, and then labeled with a fluorescent dye. The cDNA is hybridized to a gene chip containing oligonucleotide sequences representing all or a subset of genes in the organism. The amount of mRNA expressed from each gene is determined by quantitation of fluorescence intensity of the cDNA bound to each probe. The method can be adapted to compare gene expression levels in cells under different growth conditions, etc. (Fig. 5.29).

13. Cluster Analysis of Gene Expression DNA microarray data can be analyzed to identify clusters of genes with related functions that are similarly regulated under certain conditions (Fig. 5.30). As an illustration, clusters of coordinately regulated fibroblast genes that switch on or off in response to a change in media can be identified by analyzing gene expression data from several microarray experiments conducted over time. The broad functions of the clusters can be assigned (e.g., cell cycle control) based on the functions of known genes within the cluster. Cluster analysis therefore is a powerful method for deducing the functions of unknown genes.

14. Expression of Recombinant Proteins in E.coli Eukaryotic and prokaryotic proteins commonly are synthesized in E. coli for pharmaceutical and research applications. All that is required is to clone the gene of interest under the control of a strong E. coli promoter in a plasmid expression vector (Fig. 5.31). Promoters such as the lac promoter permit inducible expression by lactose, or more commonly, the synthetic lactose analog isopropylthiogalactoside (IPTG). Bacterial genes can be directly expressed in E. coli because they lack introns. cDNAs (which also lack introns) are used for expression of eukaryotic proteins in E. coli. A tag (e.g., His6) can be added to the N- or C-terminus of the protein to expedite purification (by nickel affinity chromatography).

15. Expression of Cloned Genes in Cultured Animal Cells Plasmids carrying cloned eukaryotic genes can be introduced into animal cells grown in culture by transfection. In transient transfection (Fig. 5.32a), the introduced plasmid contains a viral replication origin, which allows it to propagate for a short time until diluted out in the cells due to inefficient segregation. In stable transfection (transformation) (Fig. 5.32b), the plasmid lacks a replication origin. Thus, a selection based on the neor marker (G-418) is carried out to obtain cells in which the plasmid has integrated into the genome. Expressed proteins, which can be glycosylated, etc., are purified from transfected cells for analysis.