BLAT- Molecular and Immunological Methods.ppt
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1、BLAT: Molecular and Immunological Methods,Lyle McMillen Contact: .au,Molecular and Immunological Methods,2 basic approaches will be covered, both based on specific interactions found in vivo Nucleic acid specificity (DNA and RNA binding) Antibody recognition and interactions,Nucleic acid techniques,
2、These techniques all depend upon the specific nature of nucleic acid interactions. Namely, Adenosine forms 2 hydrogen bonds with Thymine (DNA) or Uracil (RNA). Guanine form 3 hydrogen bonds with Cytosine. Purines hydrogen bond with pyrimidines. So, A is complemented with T/U, while G is complemented
3、 with C. Interactions outside of these specific pairings are not stable. Specific nature of these pairings allows one strand of DNA or RNA to specify the nucleic acid sequence of a complementary sequence.,Base pairing,PCR a quick review,DNA is replicated and transcribed to RNA in vivo by DNA or RNA
4、polymerases, which covalently bond single nucleotides (deoxynucleoside triphosphates dA, dC, dG, and dT or dU) into a complementary sequence to the single stranded DNA template. Each strand of DNA serves as a template for the synthesis of a second, complementary strand of DNA.,PCR a quick review,The
5、 use of DNA polymerases allowed duplication of DNA when used in conjunction with a pair of primers complementary to the ends of the target DNA sequence. Unfortunately the polymerase (isolated from E. coli) degraded rapidly at high temperatures, and high temperatures were needed to denature the doubl
6、e stranded DNA produced, and allow more DNA replication. The discovery of a thermostable DNA polymerase in Thermus aquaticus allowed its inclusion in a series of repeated thermal cycles, in which the DNA was denatured to single strands, the primers annealed, and the Taq polymerase allowed to synthes
7、ise new complementary DNA. Since the discovery of Taq DNA polymerase, a number of alternative thermostable DNA polymerases have been discovered or engineered to provide different characteristics and performance in PCR.,Taq polymerase,Taq polymerase activities: Activity optimum at 75-80 C 5-3 DNA pol
8、ymerase (100 bases/second) No 3-5 exonuclease activity (ie no proofreading, and an error rate of 1 in 9000 bases) Low 5-3 exonuclease activity Polyadenylates at the 3 end, creating 3-dA overhang,Taq polymerase 94 kDa monomer,PCR a quick review,DNA sequencing a variant PCR,As DNA polymerases synthesi
9、se a second strand of DNA complementary to the sequence of the template strand, dNTPs are covalently linked to the growing polymer in a specific order. A modified PCR reaction is used to determine the order in which these nucleotides are added to the DNA polymer DNA sequencing. Addition of dideoxynu
10、cleotides (ddNTPs, lacking the 3-OH required for formation of the phosphodiester bond between 2 nucleotides) in a low concentration to the mix terminates extension of the DNA polymer at random points. A series of fragments terminated at random points in the DNA sequence are generated.,DNA sequencing
11、 a variant PCR,Key concept: Reporter molecules,DNA and RNA are fairly hard to see in a research environment, particularly in low concentrations. A variety of reporter molecules, or labels, are used to make DNA/RNA easier to detect. Fall into 2 broad categories Molecules which bind to NAs and fluores
12、ce (Dyes) used in agarose gels and some other applications. Examples: Ethidium bromide, GelRed, SYBR green Modified nucleotides which have an integral label, which are incorporated into the DNA or RNA (labels). Examples: Radioisotope (35S or 32P) labelled dNTPs, fluorescently tagged ddNTPs,DNA seque
13、ncing a variant PCR,Historically, radioactively labelled dATP was included in four separate sequencing mixes, along with one of four ddNTPs (which terminate extension when incorporated) in low concentrations. This was the Sanger, or dideoxy terminator, method, developed by Frederick Sanger and colle
14、agues in the UK in 1975. Each mix generates a population of varying length DNAs, radioactively labelled, which start with the primer sequence. These mixed populations could be separated on the basis of size (and therefore number of bases) by gel electrophoresis on a denaturing polyacrylamide-urea ge
15、l, and the different sized fragments visualized on an autoradiograph. The terminal nucleotide for each fragment was determined by which ddNTP was incorporated into the reaction.,DNA sequencing a variant PCR,A number of limitations arise from this technique. 4 datasets per DNA fragment, which need to
16、 be intregrated. Data collected manually. Short lengths of sequence data - generally 200-300 bases was as much as could be realistically achieved, although 500-800 bases were possible. Radioisotopes present a hazard to researchers and a problem for waste disposal.,Reporter molecules Fluorophores, fl
17、uorescent labels and dyes,A fluorophore is a portion of a molecule which causes that molecule to be fluorescent. Its a functional group which absorbs a specific wavelength of light and re-emits the energy at a different, specific wavelength. The wavelength absorbed is the excitation frequency, while
18、 the wavelength emitted is the emission frequency. The wavelength shift is due to a loss in energy as heat, resulting in the emission of a longer wavelength photon. This is a Stokes shift. Fluorescent labels bind specifically to the target molecule, and include a fluorophore. They bind specifically
19、to a target nucleic acid sequence. Fluorescent dyes bind to the target molecule type (eg. All DNA, or all double stranded DNA), but binding is not dependent on the target sequence. Dyes also include a fluorophore functional group.,Reporter molecules Fluorophores, fluorescent labels and dyes,Examples
20、 include: Fluorescein and the derivative Fluorescein isothiocyanate: Excitation at 494 nm, emission at 521 nm. Fluorescent dye or fluorophore used in immunohistochemistry and Fluorescent In-Situ Hybridisation (FISH) Ethidium Bromide (EtBr): A nucleic acid dye commonly used to stain DNA in electropho
21、resis. SYBR green: A nucleic acid dye, that fluoresces when intercalated in double-stranded DNA. Typically excited at one of three wavelengths (290 nm, 380 nm, and 497 nm), and emits at 520 nm. Dichlororhodamine: A range of fluorophores with different emission spectra. Used to label dNTPs 6-carboxyf
22、luorescein (6-FAM): Fluorophore used to label oligonucleotide in real time PCR.,DNA sequencing current technologies,Dichlororhodamine dyes are used to label ddNTPs in a dideoxy terminator reaction. Each ddNTP is labelled with a particular variant dye, with different emission wavelengths (i.e. Differ
23、ent colour), resulting in a single reaction generating random fragments, with each fragment labelled with a dye that corresponds to the terminal base.,Dichlororhodamine dye,DNA sequencing current technologies,These fragments can be separated on a gel or using capillary gel electrophoresis. Detection
24、 is via a laser filtered to the dye excitation wavelengths, with a corresponding emission wavelength filter to detect any fluorescence. Generates a chromatographic trace of the four emission wavelengths (corresponding to the four labelled ddNTPs).,DNA sequencing current technologies,This trace is ea
25、sily interpreted, with each peak corresponding to the terminal base on the labelled DNA fragment.,DNA sequencing current technologies,This technology presents a number of advantages compared to radioisotope labelling approaches: Single tube reaction vs 4 reactions/sample. Automated data collection,
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