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,
26、into a single data set vs manual data collection, collating 4 data sets. Generally able to read 800-1200 bases/reaction vs 200-300/reaction. No significant hazardous waste vs radioisotope waste.,A number of high throughput sequencing technologies are being developed, with the goal of sequencing mill
27、ions of bases very rapidly.,PCR end-point analysis,Conventional PCR is typically analysed by electrophoresis and visualisation of the amplicon (PCR product) on an agarose gel. Visualisation is achieved through the use of a fluorescent dye such as ethidium bromide. This occurs at the end of the PCR r
28、eaction. This is an end-point analysis.,PCR end-point analysis,PCR kinetics,Three distinct phases during a PCR reaction. Exponential phase exact doubling of product every cycle (assuming 100% efficiency). Very specific and precise. Linear phase highly variable, with reaction components starting to b
29、e consumed, products degrade, and the reaction is slowing. The extent of slowing will vary from replicate to replicate. Plateau/end-point the reaction has stopped, and no more products are being prepared. Product may begin to degrade. Final yield will vary significantly between replicates.,PCR kinet
30、ics,PCR kinetics,So, conventional PCR (via end-point analysis) is not an accurate way to quantitate the PCR template. It is also limited in its ability to quantitate different yields of amplicon using staining. It would be preferable to measure the accumulation of amplicon during the exponential pha
31、se, when the rate limiting factors are the amount of template and efficiency of amplification.,Real time PCR,Also called quantitative or kinetic PCR (but not RT-PCR, which is Reverse Transcriptase PCR). Adds a reporter molecule to a PCR reaction, allowing detection of the amplicon through the course
32、 of the PCR. This is the most important difference to conventional PCR methodologies. These reporter molecules are attached to primers, oligonucleotide probes, or the amplicon, conferring fluorescent potential on these molecules. Reporter molecules are fluorescent molecules, and are detected using a
33、 fluorescent spectrophotometer in the real time PCR platform. Two broad categories of reporter molecule they interact either specifically (labels) or non-specifically (dyes) with the amplicons nucleotide sequence. Quantitative analysis is based on detection of the amplicon during the exponential pha
34、se of the PCR. Data is presented as the thermal cycle at which the level of fluorescence reaches an arbitrary threshold, set within the exponential phase of the PCR. This is referred to as the CT value.,Real time PCR,So, how does it work? Two commonly used approaches. Double stranded DNA detection T
35、his approach utilises a fluorescent dye which specifically binds to double stranded DNA (intercalating agent) SYBR green, and later derivatives such as SYBR greener, LC green 1, SYTO 9, EVA Green. The PCR proceeds as normal, and the dye intercalates into the double stranded amplicon. The more amplic
36、on is produced, the more dye is intercalated. As these dyes intercalate, their emission intensity increases (over 100-fold for SYBR green), due to conformational changes on binding. It is worth noting that SYBR green is toxic to PCR, and is therefore used at extremely low concentrations. There are s
37、aturation dyes available that are not toxic, and can be used at higher concentrations giving stronger fluorescence.,Real time PCR,Real time PCR,The second major approach utilises hydrolysis of a specific oligonucleotide containing a fluorescent label often called the TaqMan method, but also called 5
38、 nuclease, Taq nuclease or dual-labelled probes. Taq polymerase has a 5-3 exonuclease activity. Hydrolyses DNA on the same strand as the newly synthesised DNA. The oligonucleotide probe contains 2 functional groups: a 5 fluorophore, and a 3 fluorophore (e.g. TAMRA) or non-fluorescent quencher (NFQ).
39、 Energy generated by the excitation of the 5 fluorophore is captured by the 3 quencher, and emitted as fluorescence or heat (NFQ). If a second fluorophore is the quencher, the emission wavelength is different to that of the 5 fluorophore. This process is called Fluorescence Resonance Energy Transfer
40、, or FRET. The probe anneals to the target region specifically. As the Taq polymerase synthesises DNA, it hydrolyses the probe. Cleavage of the 5 fluorophore from the rest of the probe enables it to emit fluorescence, which can be detected. The level of fluorescence detected is proportional to amoun
41、t of probe hydrolysis, and therefore the amount of amplicon synthesised.,Real time PCR,Real time PCR alternative probe strategies,There are a number of other probe strategies available, many of which are patented. Hybridisation probing entail using two probes, each labelled with a different fluoroph
42、ore (typically 6-FAM and a red fluorophore). These probes hybridise within 1-5 bases of each other on the amplicon. Excitation of the first fluorophore allows the excitation of the second fluorophore via FRET. Leads to fluorescence at the second fluorophores emission wavelength (610, 640, 670 or 705
43、 nm, depending on fluorophore), while exciting using the wavelength of the first (470 nm). Detection occurs at the end of annealing step. Once detection is complete, an increase in temperature triggers DNA polymerase activity, displacing probe and amplifying the target region. Note that when not hyb
44、ridised, the first fluorophore will emit fluorescence in its emission wavelength (530 nm for 6-FAM).,Hybridisation probes,Denaturation,Annealing,Extension,Completion,Real time PCR platforms,The real time PCR platform consists of a few basic elements Thermal cycler basically a PCR machine, usually ca
45、pable of rapid and precise variations in temperature (usually between 15 and 99 C). Excitation wavelength emitter, capable of transmitting the excitation wavelength of the fluorescent reporter to each sample. Emission detector, capable of precise quantitation of the amount of fluorescence being emit
46、ted by the sample at the fluorophores emission wavelength. Data recorder, recording the fluorescence from each sample at the end point of each thermal cycle (end of extension step).,Real time PCR platforms,There are a few major types of real time PCR platform from a range of suppliers, but they all
47、perform the same function. Most are capable of managing multiple fluorophores simultaneously, allowing multiple amplicons to be probed in a multiplex assay (well discuss this in more detail later). All are also associated with sophisticated data management and analysis software, which makes data ana
48、lysis easy, reliable and reproducible. Raw data integrity is always protected important for clinical and diagnostic applications.,Data output,Real time PCR applications,The most obvious application of real time PCR is for detection and quantitation of a specific DNA sequence. May also be used for mo
49、nitoring changes in gene expression, genotyping, or detection of genetic variations such as single nucleotide polymorphisms (SNPs).,Quantitative real time PCR,Real time PCR data is presented as CT (Cycle threshold) values, defined as the thermal cycle at which the fluorescence reaches an arbitrary t
50、hreshold. If a series of samples with known concentrations of initial template DNA is included in the assay, a linear plot of CT vs log initial template may be generated. These standards can be a known number of cells, a defined number of copies of a plasmid, or any other defined, quantifiable and reproducible number of target templates. This plot permits linear regression analysis, allowing the calculation of the copy number of any unknown target relative to the standards. The plot also indicates amplification efficiency (slope) and some indication of sensitivity (y-intercept).,
