Methods Molecular Biology 2024 (1).pptx

Methods in Molecular Biology I BCHM1250 Esther L Sabban, PhD Professor Department of Biochemistry and Molecular Biology New York Medical College, Valhalla, New York Text: Lewin’sGenes XII, Cha...

Methods in Molecular Biology I BCHM1250 Esther L Sabban, PhD Professor Department of Biochemistry and Molecular Biology New York Medical College, Valhalla, New York Text: Lewin’sGenes XII, Chapter 2 [email protected] Cloning : Genetic Engineering 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. A DNA molecular deom two (or more) different souces multiple cloning site (MCS) – A sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules. Cloning: Making an identical copy Figure 2.4: (a) A plasmid together with insert DNA (b) Restricted insert fragments and vector will be combined and (c) ligated together.  Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”. Cut up “foreign” DNA that invades the cell. Type II and III restriction enzymes cleave DNA chains at selected sites. Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage. Basics of type II Restriction Enzymes No ATP requirement. Recognition sites in double stranded DNA have a 2-fold axis of symmetry – a “palindrome”. Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.  Recognition/Cleavage Sites of Type II Restriction Enzymes Examples of Palindromes: Don't nod Dogma: I am God Cuts usually occurs at a palindromic Never odd or even sequence Too bad – I hid a boot Rats live on no evil star SmaI: produces blunt No trace; not one carton ends Was it Eliot's toilet I saw? Murder for a jar of red rum 5´ CCCGGG 3´ Some men interpret nine memos 3´ GGGCCC 5´ Campus Motto: Bottoms up, Mac Go deliver a dare, vile dog! Madam, in Eden I'm Adam EcoRI produces sticky ends Oozy rat in a sanitary zoo Ah, Satan sees Natasha Lisa Bonet ate no basil 5´ GAATTC 3´ Do geese see God? 3´ CTTAAG 5´ God saw I was dog Dennis sinned  Type II restriction enzyme nomenclature Why the funny names? EcoRI – Escherichia coli strain R, 1st enzyme BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme DpnI – Diplococcus pneumoniae, 1st enzyme Hiind3- Haemophilus influenzae, strain D, 3rd BglII – enzyme Bacillus globigii, 2nd enzyme PstI – Providencia stuartii 164, 1st enzyme KpnI – Klebsiella pneumoniae, 1st enzyme Restriction Endonucleases Cleave DNA at specific DNA sequences Cloning: Making an identical copy Figure 2.4: (a) A plasmid together with insert DNA (b) Restricted insert fragments and vector will be combined and (c) ligated together. DNA Ligase joins DNA fragments together Sticky ends that are complementary (from digests with the same or different enzymes) can be ligated together. Sticky ends that are not complementary cannot be ligated together. DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme.  Important Considerations  Safety: Will nor integrate into the genome (unless engineered specifically for that purpose) Will not auto transfer to another cell  Selection ability  High growth rate 2.3 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 2.5: The white colonies will be used to prepare DNA for further analysis. 2.4 Cloning Vectors Can Be Specialized for Different Purposes Cosmids, propages like a plasmid but uses the pckagiing mechanism if phage lamda to delive the DNA to the bacteria. Table 2.1: Cloning vectors can be based on plasmids or phages or can mimic eukaryotic chromosomes. 2.4 Cloning Vectors Can Be Specialized for Different Purposes Figure 2.6: pYac2 is a shuttle vector 2.4 Cloning Vectors Can Be Specialized for Different Purposes Cloning vectors may be bacterial plasmids, phages, cosmids, or yeast artificial chromosomes. Shuttle vectors can be propagated in more than one type of host cell. Expression vectors contain promoters that allow transcription of any cloned gene.  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. 2.5 Nucleic Acid Detection autoradiography – A method of capturing an image of radioactive materials on film. Figure 2.11: An autoradiogram of a gel prepared from the colonies described in Figure 2.5. 2.5 Nucleic Acid Detection in situ hybridization – Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography. Figure 2.12: Fluorescence in situ hybridization (FISH). Data from an illustration by Darryl Leja, National Human Genome Research Institute (www.genome.gov).  2.6 DNA Separation Techniques Gel electrophoresis separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge. Figure 2.13: DNA sizes can be determined by gel electrophoresis. Data from an illustration by Michael Blaber, Florida State University.  2.7 DNA Sequencing Classical chain termination sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at particular nucleotides. Fluorescently tagged ddNTPs and capillary gel electrophoresis allow automated, high-throughput DNA sequencing. The next generations of sequencing techniques aim to increase automation and decrease time and cost of sequencing.  DNA Sequencing Key features of DNA replication are used in DNA sequencing DNA synthesis occurs in the 5´ to 3´ direction. DNA synthesis requires a template and a primer. DNA replication is semi-conservative (one strand copied). Dideoxy NTPs block DNA synthesis A mixture of dNTPs and ddNTPs are used in DNA sequencing  Polyacrylamide gel electrophoresis can be used to visualize the results of the sequencing reaction Figure 2.16: DideoxyNTP sequencing using fluorescent tags.  2.8 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. Figure 2.17: Denaturation (a) and rapid cooling (b) of a DNA template molecule in the presence of excess primer.  Polymerase Chain Reaction (PCR) Thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions. The first round of PCR PCR increases the yield of DNA exponentially  2.9 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 2.20: Southern blot.  2.9 Blotting Methods Northern blotting is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane. Western blotting entails separation of proteins on a sodium dodecyl sulfate (SDS) gel, transfer to a nitrocellulose membrane, and detection of proteins of interest using antibodies. Figure 2.23: In a Western blot, proteins are separated by size on an SDS gel, transferred to a nitrocellulose membrane, and detected by using an antibody.  2.9 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.  2.10 DNA Microarrays DNA microarrays comprise known DNA sequences spotted or synthesized on a small chip. Figure 2.24: Gene expression arrays are used to detect the levels of all the expressed genes in an experimental sample.  2.10 DNA Microarrays Genome-wide transcription analysis 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) allows the detection of copy number changes in any DNA sequence compared between two samples.  2.11 Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) allows detection of specific protein–DNA interactions in vivo. “ChIP on chip” or “ChIP-seq” allows mapping of all the protein-binding sites for a given protein across the entire genome.  2.12 Gene Knockouts, Transgenics, and Genome Editing 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 2.26: Transfection can introduce DNA directly into the germline of animals.  2.12 Gene Knockouts, Transgenics, and Genome Editing Embryonic stem (ES) 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. Correcting Mutation with Wild type Gene  2.12 Gene Knockouts, Transgenics, and Genome Editing 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. Figure 2.28: ES cells can be used to generate mouse chimeras.  2.12 Gene Knockouts, Transgenics, and Genome Editing 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 more subtle mutations are made. 2.12 Gene Knockouts, Transgenics, and Genome Editing Figure 2.30: The Cre recombinase catalyzes a site-specific recombination between two identical lox sites, releasing the DNA between them. Tissue Specific Knockout