Chapter 3. Methods in Molecular Biology and Genetic Engineering. Chap. 3. Methods in Molecular Biology and Genetic Engineering

Transcription

1 Chapter 3 Methods in Molecular Biology and Genetic Engineering Chap. 3. Methods in Molecular Biology and Genetic Engineering 3.2 Nucleases 3.3 Cloning 3.4 Cloning vectors can be specialized for different purposes 3.5 Nucleic acid detection 3.6 DNA separation techniques 3.7 DNA sequencing 3.8 PCR and RT-PCR 3.9 Blotting methods 3.10 DNA microarrays 3.11 Chromatin immunoprecipitation 3.12 Gene knockouts and transgenics 1

2 3.1 Introduction Restriction endonuclease - An enzyme that recognizes specific short sequences of DNA and cleaves the duplex (sometimes at the target site, sometimes elsewhere, depending on type). 3.1 Introduction Cloning vector - DNA (often derived from a plasmid or a bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell. Vectors contain selectable markers and replication origins to allow identification and maintenance of the vector in the host. 2

3 3.2 Nucleases Nucleases hydrolyze an ester bond within a phosphodiester bond. Phosphatases hydrolyze the ester bond in a phosphomonoester bond. Figure 03.01: Nucleases are enzymes that degrade nucleic acids, the opposite function of polymerases. They hydrolyze, or break, an ester bond in a phosphodiester linkage. (a) phosphatase, (b) nuclease, (c) endonuclease, (d) exonuclease 3.2 Nucleases Endonuclease - Nucleases that cleave phosphoester bonds within a nucleic acid chain. They may be specific for RNA or for single-stranded or double-stranded DNA. Exonuclease- Nucleases that cleave phosphoester bonds one at a time from the end of a polynucleotide chain. They may be specific for either the 5 or 3 end of DNA or RNA. 3

4 3.2 Nucleases Restriction endonucleases can be used to cleave DNA into defined fragments: 인식하는염기의수 (4/6/8 bp), 생겨나는 ends Cohesive/sticky, blunt, 5' or 3' overhang 오른쪽그림 : 5', 3' 위치바뀜 Figure 03.02: A restriction endonuclease may cleave its recognition site and make a staggered cut or a blunt end cut. 3.2 Nucleases A map can be generated by using the overlaps between the fragments generated by different restriction enzymes. Single digestion, double digestion Figure 03.03: A restriction map is a linear sequence of sites separated by defined distances on DNA. 4

5 제한효소지도작성문제 3.3 Cloning Cloning a fragment of DNA requires a specially engineered vector. Recombinant DNA - A DNA molecule that has been created by joining together two or more molecules from different sources. 5

6 3.3 Cloning Subclone - The process of breaking a cloned fragment into smaller fragments for further cloning. MCS (multiple cloning site)- A sequence of DNA containing a series of tandem restriction endonuclease sites, used in cloning vectors for creating recombinant molecules. Unique site 3.3 Cloning 6

7 3.3 Cloning Matched sticky ends e.g. SpeI: ACTAGT XbaI: TCTAGA Figure 03.04: The final pool of this DNA will be transformed into E. coli. 3.3 Cloning Transformation - The acquisition of new genetic material by incorporation of added exogenous, nonviral DNA. Blue/white selection allows the identification of bacteria that contain the vector plasmid and vector plasmids that contain an insert. Figure 03.05: After transformation into E. coli of restricted and ligated vector plus insert DNA, the bacterial cells are plated onto agar plates containing ampicillin, IPTG (binding to repressor) and X gal. 7

8 3.4 Cloning Vectors Can Be Specialized for Different Purposes Cosmid = plasmid + phage Figure 03.06: Cloning vectors may be based on plasmids or phages or may mimic eukaryotic chromosomes. 3.4 Cloning Vectors Can Be Specialized for Different Purposes Figure 03.07: pyac2 is a cloning vector with features to allow replication and selection in both bacteria and yeast. 8

9 3.4 Cloning Vectors Can Be Specialized for Different Purposes Shuttle vectors can be propagated in more than one type of host cell. Expression vectors contain promoters that allow transcription of any cloned gene. 3.4 Cloning Vectors Can Be Specialized for Different Purposes Reporter genes can be used to measure promoter activity or tissue-specific expression. Figure 03.08_01: The graph shows the results from mammalian cells transfected with a luciferase vector driven by a minimal promoter or the promoter plus a putative enhancer. Philippe Psaila/Photo Researchers, Inc. 9

10 3.4 Cloning Vectors Can Be Specialized for Different Purposes Reporter genes can be used to measure promoter activity or tissue-specific expression. Figure 03.09: Expression of a lacz gene can be followed in the mouse by staining for β galactosidase (in blue) Figure 03.10: Since the discovery of GFP, derivatives that fluoresce in different colors have been engineered. Courtesy of Joachim Goedhart, Molecular Cytology, SILS, University of Amsterdam. Philippe Psaila/Photo Researchers, Inc. 3.4 Cloning Vectors Can Be Specialized for Different Purposes Reporter genes can be used to measure promoter activity or tissue-specific expression. Glucuronidase (GUS): X-gluc ( 기질 ) -> blue 침전물 GFP Philippe Psaila/Photo Researchers, Inc. 10

11 3.4 Cloning Vectors Can Be Specialized for Different Purposes Numerous methods exist to introduce DNA into different target cells. Figure 03.11: DNA can be released into target cells by methods that pass it across the membrane naturally, such as by means of a viral vector (1) or by encapsulating it in a liposome (2). Microinjection (3), a gene gun (4) 3.5 Nucleic Acid Detection Hybridization of a labeled nucleic acid to complementary sequences can identify specific nucleic acids. Probe : A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment. 11

12 3.5 Nucleic Acid Detection Autoradiography : A method of capturing an image of radioactive materials on film. Figure 03.12: An autoradiogram of a gel prepared from the colonies described in Figure Nucleic Acid Detection In situ hybridization - Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography. Adapted from an illustration by Darryl Leja, National Human Genome Research Institute ( Figure 03.13: Fluorescence in situ hybridization (FISH). 12

13 3.6 DNA Separation Techniques Gel electrophoresis separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge. Topoisomerase: ss break in DNA (min) Figure 03.14: DNA sizes can be determined by gel electrophoresis. Figure 03.15: Supercoiled DNAs separated by agarose gelelectrophoresis. Reproduced from W. Keller, Proc. Natl. Acad. Sci. USA 72 (1975): Photo courtesy of Walter Keller, University of Basel. Adapted from an illustration by Michael Blaber, Florida State University. 3.6 DNA Separation Techniques DNA can also be isolated using density gradient centrifugation. Figure 03.16: Gradient centrifugation separates samples based on their density. 13

14 3.7 DNA Sequencing Chain termination sequencing uses dideoxynucleotides (ddntps) to terminate DNA synthesis at particular nucleotides. Primer : A single stranded nucleic acid molecule with a 3 OH used to initiate DNA polymerase replication of a paired template strand. 3.7 DNA Sequencing Fluorescently tagged ddntps and capillary gel electrophoresis allow automated, high-throughput DNA sequencing. The next generation of sequencing techniques aim to increase automation and decrease time and cost of sequencing. Figure 03.17: DideoxyNTP sequencing using fluorescent tags. 14

15 3.8 PCR and RT-PCR Polymerase chain reaction (PCR) permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest. Forward, reverse primers Denaturation (95) -> annealing of primers (55) -> extension (72) 3.8 PCR and RT-PCR PCR depends on the use of thermostable DNA polymerases that can withstand multiple cycles of template denaturation. RT-PCR uses reverse transcriptase to convert RNA to DNA for use in a PCR reaction. - Expressional analysis - RNA isolation -> reverse transcription -> PCR 15

16 3.8 PCR and RT-PCR Real-time, or quantitative PCR detects the products of PCR amplification during their synthesis, and is more sensitive and quantitative than conventional PCR. 3.8 PCR and RT-PCR Fluorescence resonant energy transfer (FRET) - A process whereby the emission from an excited fluorophore is captured and re-emitted at a longer wavelength by a nearby second fluorophore whose excitation spectrum matches the emission frequency of the first fluorophore. 16

17 3.9 Blotting Methods Southern blotting involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe. Figure 03.21: To perform a Southern blot, DNA digested with restriction enzymes is electrophoresed to separate fragments by size. Figure 03.22: Poly(A)+ RNA can be separated from other RNAs by fractionation on an oligo(dt) column. 17

18 3.9 Blotting Methods Northern blotting is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane: gene expression analysis Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection of proteins of interest using antibodies. 3.9 Blotting Methods Western blotting Figure 03.23: In a western blot, proteins are separated by size on an SDS gel, transferred to a nitrocellulose membrane, and detected using an antibody. 18

19 3.9 Blotting Methods Epitope tag : A short peptide sequence that encodes a recognition site ( epitope ) for an antibody, typically fused to a protein of interest for detection or purification by the antibody: His tag, HA tag, Flag tag, GST tag etc 3.10 DNA Microarrays DNA microarrays comprise known DNA sequences spotted or synthesized on a small chip. Figure 03.24: Gene expression arrays are used to detect the levels of all the expressed genes in an experimental sample. 19

20 3.10 DNA Microarrays Gene expression profiling is performed using labeled cdna from experimental samples hybridized to a microarray containing sequences from all ORFs of the organism being used. SNP arrays permit genome-wide genotyping of single nucleotide polymorphisms. Array comparative genome hybridization (array-cgh) allow the detection of copy number changes in any DNA sequence compared between two samples Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) allows detection of specific protein DNA interactions in vivo. ChIP on chip allows mapping of all the proteinbinding sites for a given protein across the entire genome. 20

21 3.11 Chromatin Immunoprecipitation Figure 03.25: Chromatin immunoprecipitation detects protein DNA interactions in the native chromatin context in vivo Gene Knockouts and Transgenics Transgenics - Organisms created by introducing DNA prepared in test tubes into the germline. The DNA may be inserted into the genome or exist in an extrachromosomal structure. Figure Transfection can introduce DNA directly into the germline of animals. Photo reproduced from P. Chambon, Sci. Am. 244 (1981): Used with permission of Pierre Chambon, Institute of Genetics and Molecular and Cellular Biology, College of France. 21

22 3.12 Gene Knockouts and Transgenics ES (embryonic stem) cells that are injected into a mouse blastocyst generate descendant cells that become part of a chimeric adult mouse. When the ES cells contribute to the germline, the next generation of mice may be derived from the ES cell. Genes can be added to the mouse germline by transfecting them into ES cells before the cells are added to the blastocyst ( 배반포 : 난할기가끝난배 ) Gene Knockouts and Transgenics Figure 03.28: ES cells can be used to generate mouse chimeras, which breed true for the transfected DNA when the ES cell contributes to the germline. 22

23 3.12 Gene Knockouts and Transgenics An endogenous gene can be replaced by a transfected gene using homologous recombination. The occurrence of successful homologous recombination can be detected by using two selectable markers, one of which is incorporated with the integrated gene, the other of which is lost when recombination occurs Gene Knockouts and Transgenics Non-homologous recombination: HSV TK gene 삽입 Homologous recombination: HSV TK gene 제거 Figure 03.29: A transgene containing neo within an exon and TK downstream can be selected by resistance to G418 and loss of TK activity. 23

24 3.12 Gene Knockouts and Transgenics The Cre/lox system is widely used to make inducible knockouts and knock-ins. Knockout - A process in which a gene function is eliminated, usually by replacing most of the coding sequence with a selectable marker in vitro and transferring the altered gene to the genome by homologous recombination. Knock-in - A process similar to a knockout, but in which a gene sequence is replaced by a different gene Gene Knockouts and Transgenics Figure 03.30: The Cre recombinase catalyzes a sitespecific recombination between two identical lox sites, releasing the DNA between them. Structure from Protein Data Bank: 1OUQ. E. Ennifar et al., Nucleic Acids Res. 31 (2003): Figure 03.31: By placing the Cre recombinase under the control of a regulated promoter, it is possible to activate the excision system only in specific cells. 24

25 3 장용어 probe, RT-PCR, DNA microarray, ChIP 제목 / 학과 / 학번 / 이름 / 출처 25