Practical Course of Laboratory Methods 2017-2018
Laboratory methods description • Laboratories use a variety of methodologies to test the countless analytes that are of interest to the medical community. Understanding the method used for a test provides a broader context for understanding your test results Team of work: 2017-2018 Prof. Dr. Ahmed H. Abo Dodoma Assistant Instructor: Mona M. Moghazee Assistant Instructor: Hala Zoghly Instructor: Hader Yousry
Prof. Dr. Khaled Fahmy
Preparation of solution Lab. methods Level 3 2017-2018
Outlines: • Objective • Case study • Types of chemical materials. • Real solution vs. buffer solution. • Solution Preparation Forms • Laboratory tasks • Dilution
Objective preparation of solution • You will be able to make a solution from the solid and dilute it to a specific concentration.
Case study
5 M NaCl • Nucleic acid extraction
1% agarose gel
PCR mix preparation
• Agarose gel preparation
• dNTPs • Primers • Taq polymerase
Types of Materials
Solution
Powder
What’s different between Buffer & Solution?
• Solution: • A uniform homogeneous mixture of two or more substances. The individual substances may be present in varying amounts.
Solution
Solution component: Solute: The substance which is dissolved, or has gone into solution (typically a solid).
Solvent: The substance which does the dissolving (typically a liquid, such as water or alcohol). Must be greater than 50% of the solution.
• Buffer:
• A solution which tends to maintain a constant
pH when excess acid or base is added.
Buffer
Solution Preparation Forms Percentages %= 5% SDS 5g of SDS up to 100ml
Molar concentrations (M)= g= Mw x Mx L
Dilution= C1 V1 =C2 V2
Laboratory Tasks ✓Prepare a solution of 1.2% NaCl (%w/v) ✓Prepare 40 ml of 1.2% NaCl ✓Prepare 1000 ml of 1M NaCl
1. Dilution by Water • Cb x Vb = Ca x Va • Where C = concentration, V = volume, • b = before dilution and a = after dilution • Example: If we add 20 ml of water to 30 ml of 2% NaCl the final volume will be 50 ml and the final concentration will be 1.2% Cb x Vb = Ca? x Va Ca = Cb x Vb / Va Ca = 2 x 30 / 50 = 1.2%
2. Dilution By mixing different concentrations • Cf x Vf = (C1 x V1) + (C2 x V2) • Where C = concentration, V = volume, • f = final solution, 1 = 1st solution and 2 = 2nd solution • Example: if we add 20 ml of 1% NaCl to 30 ml of 2% NaCl the final volume will be 50 ml and the final concentration will be 1.6% • Cf? x Vf = C1 x V1 + C2 x V2 • Cf = [C1 x V1 + C2 x V2] / Vf • Cf = [1 x 20 + 2 x 30] / 50 = 1.6%
3. Dilution By mixing different solutions • Cb x Vb = Ca x Va • Where C = concentration, V = volume, • b = before mixing and a = after mixing • Example: if we add 20 ml of 1% KCl to 30 ml of 2% NaCl the final volume will be 50 ml and the final concentration of NaCl will be 1.2% and for KCl will be 0.4% Cb x Vb = Ca x Va Ca = Cb x Vb / Va Ca [for NaCl] = 2 x 30 / 50 = 1.2% Ca [for KCl] = 1 x 20 / 50 = 0.4%
Laboratory Tasks ✓Prepare a solution of 1.2% NaCl (%w/v) ✓Prepare 40 ml of 1.2% NaCl ✓Prepare 1000 ml of 1 M NaCl ✓Prepare 0.5 % NaCL from 1% NaCl solution
How to make a solution?
Researcher Arnold Beckman
Researcher (Arnold Beckman ) Chemist, Scientist, Educator (1900–2004) • Arnold Beckman worked as a chemist and professor before creating the "acidimeter," meant to measure acidity levels and thus becoming the first pH meter. Beckman founded his own business, Beckman Instruments, and invented a number of additional items, including the Beckman Spectrophotometer.
Devices & laboratory equipment
Explain, How the Devices work and precautions of these devices that use in preparation of solution?
10
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Explain, How this Device work and the precautions of this?
10
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Explain, How this Device work and the precautions of this?
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Blotting technology
Lab. Methods
Letter Lab. methods Level 3 2017-2018
1 S
Calculation Tool Researcher Technology /Technique Device Sumiton = Total=
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Description
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Tools “Syringe” A tool used for injecting or withdrawing liquids.
Explain technique that works on this tool? 10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
Researcher (Susumu Tonegawa) • Famous As: Immunologist, Molecular Biologist.
• In 1987, he was awarded the Nobel Prize in Physiology or Medicine "for his discovery of the genetic principle for generation of antibody diversity."
Mention another researcher begins with a letter S • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
10
Technology or Technique or Terms “Southern blotting”
Markers in biology: (genetics) A gene or DNA sequence with known physical location, and whose pattern of inheritance can be followed.
Molecular markers • Based on hybridization • Based on PCR generation
Biochemical markers • Isozyme • Protein banding pattern
Southern blotting definition
• The southern blotting principle is Hybridization, Which is the process of forming a double stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA.
Southern blotting steps 1. 2. 3. 4. 5. 6. 7. 8.
Extract and purify DNA from cells. DNA is restricted with enzymes. Separated by electrophoresis. Denature DNA. Transfer to nitrocellulose paper. Add labeled probe for hybridization to take place. Wash off unbound probe. Autoradiograph.
Southern blotting steps
Southern blotting Advantages • Effective way to detect a specific DNA sequence in large, complex sample of DNA. • Can be used to quantify the amount of the present DNA. • Cheaper than DNA sequencing.
Dis-advantages • More expensive than most other tests. • Complex and labor intensive.
Southern blotting applications
1
2
3
4
To identify specific
To Isolate desired
Identify mutations,
In DNA
DNA in a DNA
DNA for construction
deletions, and gene
fingerprinting
sample.
of rDNA.
rearrangements.
In the light of your previous study, • what are the tools and equipment needed to do the southern blotting technique? • ………………………………………… ………………………………………… …………………………………………
10
Device Spectrophotometer spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry is an important technique used in many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses.
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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Letter N Lab. methods Level 3 2017-2018
Calculation Tool Researcher Technology /Technique Device Sumiton = Total=
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Description
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Tools “Needle of inculcation� It is a laboratory equipment used in the field of microbiology to transfer and inoculate living microorganisms
Explain technique that works on this tool? 10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
Researcher (Norman Borlaug) • Famous As: Father Of The Green Revolution • Nationality: America
• Over the course of his work he successfully developed disease resistant, high yielding wheat varieties which when combined with modern agricultural production techniques could dramatically change the way farming was done.
Mention another researcher begins with a letter N • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Technology or Technique or Terms “Northern blotting”
Northern blotting definition
• The northern blotting principle is Hybridization, Which is the process of forming a double stranded DNA-RNA-hybrid molecule between a singlestranded RNA probe and a single-stranded target RNA.
Northern blotting steps 1. Extract and purify mRNA from cells. 2. Separated by electrophoresis. 3. this gel is immersed in depurination buffer for 5-10 minute then washed with water. 4. Transfer to aminobenzyloxymethyl filter paper. 5. After transfer, the membrane is baked at 80áľ’c 6. Add labeled probe for hybridization to take place. 7. Wash off unbound probe. 8. Autoradiograph.
Northern blotting steps
Southern blotting vs. Northern blotting Southern blotting
Northern blotting
Northern blotting applications
To detect specific mRNA in a sample.
To screen recombinants by detecting the mRNA produced by the transgene
Diseases diagnosis.
Gene expression studies
In the light of your previous study, • what are the tools and equipment needed to do the Nouthern blotting technique? • ………………………………………… ………………………………………… …………………………………………
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Device Numerical pH meter Fast, Accurate, and Auto Calibrated instrument used for adjustment of pH
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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Letter W lab methods level 3 2017-2018
Calculation Tool Researcher Technology /Technique Device Sumiton = Total=
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Description
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Tool Wire gauze It is a sheet of thin metal. wire gauze sits on the iron ring to provide a place to stand a beaker. on older wire gauze, the white material was asbestos currently it is a ceramic.
Explain technique that works on this tool? 10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
Researcher William Harvey 1578-1657
William Harvey was a 17th-century. British physician who became the first to document an understanding of blood circulation
Harvey's research was furthered through the dissection of animals.
William Harvey 1578-1657
He published his theories in a book entitled (Anatomical Study of the Motion of the Heart and of the Blood in Animals), where he explained how the heart propelled the blood in a circular course through the body.
William Harvey 1578-1657
Harvey was also the first to suggest that humans and other mammals reproduced via the fertilization of an egg by sperm. It took a further two centuries before a mammalian egg was finally observed, but nonetheless Harvey's theory won credibility during his lifetime
Mention another researcher begins with a letter W? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Technology or Technique or Terms “Western blotting”
Western blotting (protein blotting or immunoblotting) • Western blotting can produce qualitative and semiquantitative data about the protein of interest. • It is an important technique used in cell and molecular biology. • It enables the researchers to identify the specific protein from mixture of proteins extracted from cells as well as evaluation of their size and amount. • The SDS PAGE technique is prerequisite for western blotting.
What’s mean the SDS PAGE? SDSPolyAcrylamide Gel Electrophoresis
Western blotting steps
Tissue preparation
Gel electrophoresis
Transfer
Blocking
Detection (two step , one step)
Western blotting steps
Western blotting steps •Proteins are extracted from the sample •Proteins are separated by their sizes using polyacrylamide gel electrophoresis (SDS-PAGE) •Separated molecules are transferred into a *PVDF membrane or nitrocellulose membrane by electroporation •The membrane is blocked for non specific binding with the antibodies *PolyVinylidene DiFluoride (PVDF) Membranes
Western blotting steps •Transferred proteins are bound with primary antibody (enzyme labeled antibodies). •The membrane is washed to remove nonspecifically bound primary antibodies •Bound antibodies are detected by adding a substrate and
detecting the colored precipitate formed.
Western blotting Uses ➢ It is most sensitive and specific test for determining size and amount of protein present in any material. ➢ The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. ➢ A western blot is also used as the definitive test for Creutzfeldt-Jakob Disease, Lyme disease, Hepatitis B infection and HSV-2 (Herpes Type 2) infection.
Advantages & Disadvantages
Advantages
Disadvantages
Sensitivity
High cost
Because of its ability to detect as little as 0.1 ng of protein in a sample, the technique can theoretically serve as an effective early diagnostic tool, sensing even the slightest immunogenic response from a virus or bacteria in a patient sample.
Technical Demand
In the light of your previous study, • what are the tools and equipment needed to do the western blotting technique? • ………………………………………… ………………………………………… …………………………………………
10
Device Water bath A water bath is a device that maintains water at a constant
temperature. It is used in the Biotechnology laboratories
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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References • http://www.bbc.co.uk/history/historic_figures/harvey_william.shtml • https://www.slideshare.net/136659145/western-blot2563509?from_action=save • http://www.microeguide.com/equipment/water_bath.asp • https://www.slideshare.net/136659145/western-blot2563509?from_action=save
Gene expression techniques
Letter D Lab. methods ( ) Level 3 2017-2018
Calculation Tool Researcher Technology /Technique Device Sumiton = Total=
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Description
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Tools “Desiccator” • Desiccators create and maintain dry environments to ensure sample stability. • Using absorbing beads, and other desiccants, desiccator cabinets or apparatuses quickly absorb moisture, guaranteeing sensitive materials do not react to surroundings prior to testing.
Explain technique that works on this tool? 10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
David Baltimore
Researcher (David Baltimore) • Famous As: Virologist • He is an American biologist who won a share of the 1975 Nobel Prize in Physiology or Medicine. As a researcher, he has made tremendous contributions to immunology, virology, cancer research, biotechnology, and recombinant DNA research. • he performed pioneering research on animal virology. Later in his career, he independently discovered reverse transcriptase, an enzyme that synthesizes DNA from RNA. • And researched on interaction between viruses and the genetic material of the cell.
Mention another researcher begins with a letter D • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Technology or Technique or Terms “Differential display PCR” “DDRT-PCR or DD-PCR”
Differential display definition
It is a laboratory technique that allows a researcher to compare and identify changes
in
gene
expression
at
the mRNA level between two or more eukaryotic cell samples
Introduction • Differential display is a rapid method which can be applied to detect changes in the transcriptome in response to environmental and temporal factors.
• Identification of mRNA, SnRNA and miRNA.
Principle of Differential display Differential display relies on the use of two distinct primers: an anchor primer and a second primer with an arbitrarily chosen sequence. Anchor primers are used in both the reverse transcription reactions and in the subsequent PCR amplification while the second, arbitrary primer is used only in the PCR reaction.
DD-RT PCR technique • Anchored primer • Arbitrary primer
Sequi-Gen GT System component
Troubleshooting Device: 1. Washing the two glass by 10% NaoH “leave it overnight for removing all residual to No form bubbles” then double distal H2O finally Ethanol absolute. 2. Machine composition: you should put binding solution on glass “one of them”. Putting sigma coat on the other glass
Advantages
Differential display
• the rapidity and sensitivity of the assay. • Does not require any prior knowledge of the genome. • Reproducibility. • Speed and Expense.
Dis-advantages • Not easily automated or scaled-up. • Non-specific and inefficient amplification. • Generate positive results unwanted.
Differential display applications
1 Identification of transcripts that are differentially regulated.
2 Essential step prior to carry out whole transcriptome sequencing
3 Validation of RNA transcripts isolated from a whole transcriptome library
In the light of your previous study, • what are the tools and equipment needed to do the Differential display technique? • ………………………………………… ………………………………………… …………………………………………
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Device Distilled Water Machine
Distilled Water Machine • Equipment used for water purification and distillation includes Deionized (DI) Water Systems, water distillers, reagent-grade water systems, and laboratory filters. DI and distilled water are the most common types of purified water used in the lab, but techniques also used to produce high-purity water include: such as; Carbon filtration.
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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Letter I Lab. methods ( ) Level 3 2017-2018
Calculation Tool Researcher Technology /Technique Device Sumiton = Total=
Name
Description
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Tools “iris scissors” • Iris scissors are a type of scissors with short blades that was originally developed for ophthalmic surgery. • Iris scissors are also available in the crafting market and are sometimes used for the production of fabric-related goods.
Explain technique that works on this tool? 10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
Isaac Asimov
Researcher (Isaac Asimov) • Famous As: Writer, Professor • Isaac Asimov is best known as the most successful writer of science fiction and popular science books. • Asimov opened the doors for the new age of science fiction writing which the world had never tasted before him. Asimov is credited with having edited over 500 books.
Researcher (Isaac Asimov) • Asimov was a brilliant professor of biochemistry at Boston University. Besides being a prolific writer, Asimov was also an
integral part of (President) the American Humanist Association. Asimov is also known for his work as a civilian at the
Philadelphia Navy Yard's Naval Air Experimental Station during the World War II. “Robotics” was a term coined by Asimov
which went on to become a branch of technology.
Mention another researcher begins with a letter D • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Technology or Technique or Terms In Situ Hybridization “ISH”
What’s mean in situ, in molecular and cell biology? In molecular and cell biology, • in situ It means "locally", "on site", "on the premises" or "in place" to describe an event where it takes place, and is used in many different contexts. For example, in fields such as biology, in situ may describe the way a measurement is taken, that is, in the same place the phenomenon is occurring without isolating it from other systems or altering the original conditions of the test..
Introduction •In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole
mount ISH), in cells, and in circulating tumor cells (CTCs). •It obtain temporal and spatial information about gene expression
and genetic loci
Scientific Basis of ISH • Double-stranded DNA denatures on heating to single-stranded DNA. On cooling, the single-stranded DNA reanneals with its complementary sequence into double-stranded DNA. • labeled fragment of a DNA sequence (a DNA probe) is denatured and added to denatured nuclei or chromosomes on a routine, air-dried interphase preparation during the process of reannealing, some of the labeled DNA will hybridize to its complementary sequence in the chromosomal DNA
ISH principle 1. Denaturation
annealing
2. Labelling
examination
Types of In Situ Hybridization • While the basic workflow of ISH is similar to that of blot hybridizations. the nucleic acid probe is synthesized, labeled, purified, and annealed with the specific target—the difference is the greater amount of information gained by visualizing the results within the tissue. • There are two basic ways to visualize your RNA and DNA targets in situ fluorescence (FISH) and chromogenic (CISH) detection.
Types of In Situ Hybridization Instrument/ visualization method
Technique
Primary advantage
Primary application
Bright-field microscopy
Ability to view the CISH signal and tissue morphology simultaneously
Molecular pathology diagnostics
DNAFISH
Fluorescence microscopy
Multiplexable: visualize multiple targets in the same sample
Gene presence, copy number, and location; mutation analysis
RNAFISH
Multiplexable: visualize Fluorescence microscopy, HCS, and multiple targets in the same sample flow cytometry
CISH
FISH
Gene expression, RNA temporal and spatial localization
Multicolor FISH
1. Fluorescence In Situ Hybridization
2. Chromogenic In Situ Hybridization (CISH)
Chromogenic In Situ Hybridizati on (CISH) •Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization
Different between direct & indirect ISH http://pubs.rsc.org/en/content/articlehtml/2014/ra/c3ra45401k
Directly labelled ISH probe • Principle of in situ hybridization. This figure represents the principles of ISH using either a directly- or indirectly- labelled method. In directly-labelled ISH, the DNA probe is directly conjugated to a fluorophore (yellow), as occurs in FISH.
Indirectly labelled ISH probe • In indirectly-labelled ISH, the DNA probe is conjugated to a hapten (pink). During the denaturation step of the ISH protocol, the DNA probe hybridizes to the gene of interest. Hapten-conjugated probes are detected either with a chromogen-linked antibody (red) (CISH), or with a fluorescentlylabelled antibody.
3. in situ PCR • in situ PCR is a method in which the polymerase chain reaction actually takes place in the cell on a slide, and the product can be visualized in the same way as in traditional in situ hybridization.
3. in situ amplification "In situ PCR” • In situ PCR has mainly been used to identify DNA sequences that are not easy to detect using standard in situ hybridization. These sequences include human single‐copy genes, chromosomal translocations, and rearranged cellular genes. • In situ PCR is also used for mapping genomic sequences that have a low copy number in metaphase chromosomes. However, in situ PCR has multiple problems, including low efficiency of amplification and poor reproducibility.
Catalyzed reporter deposition (CARD)
4. Catalyzed reporter deposition (CARD) • This method involves deposition of activated biotinylated tyramine onto electron rich moieties (e.g., tyrosine and phenylalanine) at or close to the site of horseradish peroxidase (HRP). • When using HRP-labeled probes fluorescent staining results from a secondary incubation with fluorescently labeled tyramine. • The specifically bound peroxidase molecules catalyze the deposition of these labeled reporter compounds within cells targeted by the HRP-tagged probe.
In Situ Hybridization steps:
https://greenfluorescentblog.wordpress.com/2013/02/21/rnascope-a-novel-fish-in-the-sea/
In Situ Hybridization steps: 1. Preparation of Tissue • Treatment with proteases (proteinase K is the most common) • Acetylation of sections • Optimization of tissue processing, including fixation and storage
2. In situ hybridization probes: • Double-stranded DNA (dsDNA) probes • Single-stranded DNA (ssDNA) probes • RNA probes (riboprobes)
In Situ Hybridization steps: 3. Labeling techniques: • Radioactive probe 32P, 125I, 35S, or 3H • Non-radioactive labels • biotin • digoxigenin • fluorescent dye (FISH)
In Situ Hybridization example • In situ hybridization of wild type Drosophila embryos at different developmental stages for the RNA from a gene called hunchback.
Hybridization
In Situ
Advantages • Maximum use of tissue that is difficult to obtain (e.g., embryos and clinical biopsies). • Hundreds of different hybridizations can be performed on the same tissue. • Libraries of tissues can be formed and stored in the freezer for future use.
Dis-advantages • Difficulty in identifying targets that have low DNA and RNA copies. However, approaches are continually being developed to improve the sensitivity of in situ hybridization
Applications of
In Situ Hybridization
• Microbiology • Pathology • Developmental biology • Karyotyping and phylogenetic analysis • Physical mapping
In the light of your previous study, • what are the tools and equipment needed to do the In Situ Hybridization technique? • ………………………………………… ………………………………………… …………………………………………
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Device Incubator
Incubator Device •In biology, an incubator is a device used to grow and maintain microbiological cultures or cell cultures. •The incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside. •Incubators are essential for a lot of experimental work in cell biology, microbiology and molecular biology and are used to culture both bacterial as well as eukaryotic cells.
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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Letter M Lab. Methods Level 3 2017-2018
Calculation Name
Description
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Sumiton
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Tool Researcher Technology /Technique Device
Discussion
Tools Magnetic Stir Bars
•A stir bar is the magnetic bar placed within the liquid which provides the stirring action. The stir bar's motion is driven by another rotating magnet or assembly of electromagnets in the stirrer device, beneath the vessel containing the liquid
Mention another Tools begins with a letter M • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Researcher Marguerite Vogt
Researcher Marguerite Vogt Marguerite Vogt was a German-born, American cancer biologist and virologist best known for her research on Polio
And Cancer at the ‘Salk Institute for Biological Studies’. She collaborated
with
Nobel
Prize-winning
scientist
Renato
Dulbecco to analyze the way polio virus develops plagues in cell
cultures,
a
discovery
that
development of a polio vaccine. https://www.thefamouspeople.com/profiles/marguerite-vogt-7505.php
eventually
aided
in
the
Mention another researcher begins with a letter M • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Micro array
“cDNA microarray” “Spotted Array”
Technology or Technique or Terms “Micro array” • The technique of microarrays came into use in the late 1990s. Many cDNA clones were available from selected model organisms. • Many cDNAs can be spotted onto an array that is used to determine changes in the level of expression of all cDNAs in a comparison of two samples.
Introducing DNA Microarray •A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA
fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically
spotted (less than 1 mm) to that location. Through the use of highly accurate robotic spotters, over 30,000 spots can be
placed on one slide.
Microarray uses • DNA microarrays can be used to detect DNA (as in comparative genomic hybridization), or detect RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins. • The process of measuring gene expression via cDNA is
called expression analysis or expression profiling.
Affymetrix “oligonucleotides chips”
Microarray chip • A microarray is a glass microscope slide
spots,
containing
either
thousands
cDNA
clones
of
or
synthetic oligonucleotides.
• In the example below, each spot is a single exon from a known gene.
Microarray basic
The Experiment • comparison the mRNAs of control cells to mRNAs from cells exposed to high temperature
Pipeline steps of Experiment: RNA extraction
cDNA synthesis
Labeling
Data anaylsis
Screening by laser light
Hybridization on Chip
The Experiment steps
The Experiment results:
Aim of assay • The assay is actually semiquantitative, with a full range of colors between red and green showing the relative abundance of a particular cDNA in the two samples
Cluster analysis • In this example, green is an increase in expression in the tumor relative to control, while red is a decrease. This is the usual convention
Microarray uses: • Microarray analysis can also be used to identity new tumor subtypes based on patterns of gene expression
Microarray uses: • Microarray analysis can be used to determine the outcome of a standard course of treatment.
Microarray uses: • Differences in the patterns of gene expression in tumors that appear to be identical might also be used to identify drugs that would be useful in a particular case
1 Gene expression profiling.
2
3
Chromatin immunopreci pitation "CHIP"
SNP detection
Microarray applications
Microarray Advantages • Fast and easy to obtain results • Different parts of DNA can be used to study gene expression. • Huge step closer to discovering cures for diseases and cancer
Dis-advantages 1.Correlations in results do not mean causation 2.It will the findings lead to unethical medical procedures 3.Very little knowledge is available about many genes
In the light of your previous study, • Mention, Different method to determination gene expression? • …………………………… …………………………… …………………………… …………………………… 10 ……………………
Device Magnetic stirrer • A magnetic stirrer or magnetic mixer is a laboratory device that employs a rotating magnetic field to cause a stir bar (also called "flea") immersed in a liquid to spin very quickly, thus stirring it. • Magnetic stirrers are often used in chemistry and biology
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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Letter Lab. methods Level 3 2017-2018
2 S
Calculation Name
Description
Picture
Sumiton
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-------/ 40 Researcher
-------/ 20 Technology /Technique
------/100 Device
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Tool Researcher Technology /Technique Device
Discussion
• Laboratory double spatulas in stainless steel to take a solid (e.g. powder). • Two flat spatulas and a spoon spatula.
Tools
“Spatulas-lab”
Do you know different use for these?
10
• ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………
Researcher (Salvador Edward Luria) • Famous As: Microbiologist • Who jointly won the Nobel Prize in Physiology or Medicine in 1969 with Max Delbrück and Alfred Hershey, for their discoveries on the replication mechanism and the genetic structure of viruses.
Mention another researcher begins with a letter S • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………..… ………….………………………………………….… ……………………………………………………..… ………………………………………………………..
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Technology or Technique or Terms “Sequencing”
Basic Methods of Sequencing Maxam & Gilbert Sequencing “Chemically method” Sanger Sequencing Enzymatically method
1. The Chemical Sequencing Method Maxam & Gilbert
Maxam & Gilbert Sequencing • This method based on chemical modification of DNA and subsequent cleavage at specific bases. • Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. • Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T).
Maxam & Gilbert Sequencing Preparation Tube
• this method is based on nucleobasespecific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides
Maxam & Gilbert Sequencing Migration
Maxam & Gilbert Sequencing Reading Seq.
Movie
https://www.youtube.com/watch?v=lqWZ-duHfu8
Sanger Sequencing Method
“The chain-termination Method�
Sanger Sequencing • The target DNA is copied many times, making fragments of different lengths. Fluorescent “chain terminator” nucleotides mark the ends of the fragments and allow the sequence to be determined. • This separates the fragments by size, the smaller ones traveling the furthest. Each of the four reactions are run in one of four individual lanes, and the DNA bands are then visualized by autoradiography or UV light. Then the DNA sequence can be directly read on the X-ray film or gel, by reading up the lanes.
Sanger Sequencing Preparation Tube • The classical chainterminator method requires (DNA+ primer+ dNTPs) and all modified dideoxynucleotidetrip hosphates (ddATP, ddGTP, ddCTP, and ddTTP)
Sanger Sequencing in vitro tube • DNA fragments are labelled with a radioactive or fluorescent tag on the primer, in the new DNA strand with a labeled dNTP, or with a labeled ddNTP.
Sanger Sequencing Reading Seq.
Manually
Automatically
Sanger Sequencing Reading Seq.
Maxam & Gilbert Vs. sanger seq.
Sanger Sequencing More recently, higher volume Sanger sequencing has been supplanted by "Next-Gen" sequencing methods, especially for large-scale, automated genome analyses.
Applications of sequencing Molecular biology
Metagenomics
Evolutionary biology
Medicine
Forensics
Applications of sequencing 1. Molecular biology • Molecular biology: Sequencing is used to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, and identify potential drug targets.
Applications of sequencing 2. Evolutionary biology • Evolutionary biology: Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in evolutionary biology to study how different organisms are related and how they evolved.
Applications of sequencing 3. Metagenomics • Metagenomics: The field of metagenomics involves identification of organisms present in a body of water, sewage, dirt, debris filtered from the air, or swab samples from organisms.
Applications of sequencing 3. Metagenomics • Metagenomics: Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology, microbiology, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example.
Applications of sequencing 4. Medicine • Medicine: Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. This is a form of genetic testing, though some genetic tests may not involve DNA sequencing.
In the light of your previous study, • what are the tools and equipment needed to conduct the experiment transformation? • ………………………………………… ………………………………………… …………………………………………
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Device scale • balance designed to measure small mass in the submilligram range.
Explain the technique that works on this device? • ……………………………………………………… ……………………………………………………… ……………………………………………………… ……………………………………………………… ………………………………………………………
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