CELL BIOLOGY - CLUTCH CH TECHNIQUES IN CELL BIOLOGY.

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2 CONCEPT: LIGHT MICROSCOPE A light microscope uses light waves and magnification to view specimens Can be used to visualize transparent, and some cellular components - Can visualize 10-20µm in diameter - Limit of resolution is the distance that you can tell two objects apart is 0.2µm - Resolving power is the ability to see fine details of a structure (smaller resolution = greater resolving) Light microscopes can be to increase visualization of components - Bright field microscopy is when light passes directly through a specimen, but it needs to be fixed - Phase contrast microscopy improves the contrast of live cells - Differential inference microscopy can be used to visualize live cells - Fluorescence microscopy uses fluorescence to detect specific proteins or organelles - FRAP studies movement of fluorescently labeled proteins - FRET visualizes two interacting proteins within a cell EXAMPLE: Page 2

3 PRACTICE: 1. Which of the following terms describes the smallest distance that you can tell two objects apart? a. Resolving power b. Limit of resolution c. Convex power d. Phase limit 2. Which type of fluorescence microscopy can visualize two interacting proteins in a cell? a. FRAP b. FRET c. FREP d. FRAT Page 3

4 CONCEPT: ELECTRON MICROSCOPY Electron microscopy uses electrons to cells Improved limit of resolution, up to 0.002nm (100,000x greater than light microscope) To perform electron microscopy, the samples have the be properly - Specimens need to be preserved using special chemicals - Specimens need to be sliced extremely thin (1/200 thickness of a cell) There are many ways to process samples - One way to process the sample is to use immunogold staining - Immunogold staining labels specimens with electron dense gold (via antibodies) - Metal shadowing is used by coating specimen with metal at one angle, creating electron dense shadows There are two main of electron microscopy - Transmission electron microscopy which shoots electrons through the samples - Scanning electron microscopy which scans electrons over the specimen s surface. EXAMPLE: Electron microscopy image of mitochondria Page 4

5 PRACTICE: 1. True or False: Light microscopy has an improved limit of resolution compared to electron microscopy. a. True b. False 2. Which of the following types of microscopy works by shooting electrons through the specimen? a. Light microscopy b. Transmission electron microscopy c. Scanning electron microscopy d. FRAP floruences microscopy Page 5

6 CONCEPT: RADIOISOTOPES Radioactive isotopes are atoms where the nucleus is unstable, and can emit There are three types of radiation that can be released - Emission of alpha particles results in loss of two protons and two neutrons - Emission of beta particles results in the loss of an electron - Emission of gamma particles results in the loss of photons, changing the energy state of nucleus - Autoradiography is a technique that detects isotopes in biological materials (cell, gel, filter) Radioactive isotopes can be attached onto. - Determine quantity of molecules in a cell - Determine location of molecules in a cell - Follow the movement of a molecule in a cell over time or in response to an environmental stimulus EXAMPLE: Radioisotope labeled molecules visualized on a gel Page 6

7 PRACTICE: 1. Which of the following types of radiation results in the loss of an electron? a. Emission of alpha particles b. Emission of beta particles c. Emission of gamma particles 2. Which of the following characteristics can a radioisotope NOT determine? a. The quantity of molecules in a cell b. The location of molecules in the cell c. The size of a single molecule in the cell d. The movement of a molecule in the cell Page 7

8 CONCEPT: CELL CULTURE Cells can be cultured in a setting (Began in 1907) Primary cultures are derived directly from tissues - Take tissue, disrupt the ECM, and dissect cells from thin tissue slices - Unfortunately, they don t live very long and are difficult to keep alive Secondary cultures are cells that are derived from other cultured cells - Cell lines are cells that have undergone genetic modifications to allow them to grow indefinitely - Eventually each culture will die after divisions but you can freeze them at early cycles Cells need a lot of when grown in a laboratory - Need certain nutrients from media solutions - Need to be grown in flat dishes, or in a 3D environment with fake ECM - Need to be maintained at certain temperatures with certain gas exchanges Cell culture provides certain benefits to scientists - Provides homogenous population of cells to work with - More convenient to work in a lab Research done using is called: - In vitro by most everyone because it doesn t happen in living organisms - In vivo by biochemists, because it does happen in living cells EXAMPLE: Page 8

9 PRACTICE: 1. Which cell types are derived from primary tissue? a. Primary cell cultures b. Secondary cell cultures c. Tertiary cell cultures d. Cell lines 2. In vitro can describe experiments happening in all but which of the following? a. In a tube b. In cells c. In living organisms Page 9

10 CONCEPT: ISOLATION AND PURFICATION OF PROTEINS There are a variety of techniques that scientists use to obtain for research. Protein purification allows for isolation of a single protein - Protein is grown in bacterial cultures and isolated - Fractionalization separates different proteins into individual fractions based on properties (size, charge) - Proteins can be tagged to give them a certain property that makes them easier to purify Chromatography allows for separation of proteins via certain properties - Column chromatography separates mixture of proteins by running them through a porus mixture (Size) - Affinity chromatography sorts proteins based on interactions with other proteins - Gel filtration chromatography separates proteins based on size Gel electrophoresis is used to separate proteins based on a charge to mass ratio Once a purified protein is obtained, the proteins can be in isolation EXAMPLE: Column Chromatography Page 10

11 PRACTICE: 1. Which of the following methods allow for the isolation of a single protein? a. Protein purification b. Affinity chromatography c. Gel Electrophoresis d. Column chromatography 2. Which of the following methods separates proteins by a charge to mass ratio? a. Protein purification b. Affinity chromatography c. Gel Electrophoresis d. Column chromatography Page 11

12 CONCEPT: STUDYING PROTEINS Scientists use a variety of to study protein structure and function SDS Polyacrylamide gel-electrophoresis (SDS-PAGE) is used to detect proteins - Protein solutions are run through a polyacrylamide matrix (contains adjustable pores for migration) - Proteins unfold when attached to negatively charged SDS, and therefore migrate in response to + charge - The gel is then transferred onto nitrocellulose paper via a second electric field - The membrane is then immunoblotted with antibodies to identify if proteins are present 2D Gel electrophoresis can separate up to 2000 proteins on one membrane - Proteins are separated by intrinsic charges and ph EXAMPLE: SDS PAGE A B Mass spectroscopy is used to identify unknown proteins - Samples peptides via a mass to charge ratio - Provides sequences of proteins from unknown protein solutions Nuclear magnetic resonance spectroscopy (NMR) is used to analyze structures of proteins in solution Yeast-two hybrid system is able to examine if two proteins interact inside a living organisms Page 12

13 - Two proteins of interest (called bait or prey ) are fused to transcription factors - If bait and prey proteins interact, then the transcription factors interact - The interaction is created so that when the bait and prey interact it activates transcription of a gene EXAMPLE: Yeast two hybrid system Bait Prey Transcription Promoter Reporter Gene PRACTICE: 1. Which method would be best to use if you wanted to identify unknown proteins? a. SDS-PAGE b. Mass Spec c. NMR d. Yeast two-hybrid test Page 13

14 2. Which method would be best to use if you wanted to analyze the structure of a protein? a. SDS-PAGE b. Mass Spec c. NMR d. Yeast two-hybrid test Page 14

15 CONCEPT: NUCLEIC ACID HYBRIDIZATION Hybridization takes advantage of the fact that complementary nucleic acid strands will form Southern blotting detects specific genes in cellular DNA - DNA from a cell is separated by gel electrophoresis - The DNA is transferred to a filter paper - A DNA probe complementary to a sequence of interest is incubated with the filter - The probe is fixed with a fluorescent or radioactive molecule for detection Northern blotting detects specific RNA sequences EXAMPLE: Southern Blotting A 5kb B 2kb 3kb A B 5kb 3kb 2kb Page 15

16 PRACTICE: 1. Which method is used to detect RNA sequences? a. Northern blotting b. Southern blotting c. Western blotting d. Eastern blotting Page 16

17 CONCEPT: DNA CLONING DNA cloning is the process of assembling DNA molecules for experimental use (genetic engineering) 1. Obtain DNA that you want to manipulate - DNA extractions, Polymerase Chain Reaction of specific sequences, 2. Digest with restriction endonucleases (enzymes) which cut DNA at specific sequences 3. Paste the desired fragment into a vector - A vector (plasmid) is a small circular DNA molecule that replicates independently of the organism - DNA ligase is the enzyme responsible for sealing two DNA fragments together 4. Place the vector containing the DNA sequence into an organism or cell (usually E.coli) - In E. coli this allows for large quantities of a specific DNA sequence EXAMPLE: Restriction Enzyme DNA Vector E. coli Page 17

18 PRACTICE: 1. Which of the following shows the correct order of DNA cloning steps? a. DNA extraction à Digestion à Place DNA into E. coli à DNA ligase b. DNA ligase à Digestion à DNA extraction à Place DNA into E. coli c. DNA extraction à Digestion à DNA ligase à Place DNA into E. coli d. Digestion à DNA extraction à DNA ligase à Place DNA into E. coli Page 18

19 CONCEPT: POLYMERASE CHAIN REACTION Polymerase chain reaction (PCR) is a method for amplifying DNA Includes multiple of: 1. Exposing the DNA to high temperatures to separate the two strands 2. Lowering the temperature to allow for complementary primers to bind to initiate replication 3. DNA polymerase begins replicating the DNA Important for creating billions of copies of a desired nucleotide sequence which can be used in: - Diagnostics and forensics applications - Comparing DNA molecules - Testing for presence of a specific DNA sequence - Quantifying the amount of DNA (qpcr) or RNA (RT-PCR) in a sample EXAMPLE: Cycle 1 Cycle 2 Page 19

20 PRACTICE 1. How many copies of DNA exist after 4 cycles of PCR? a. 4 b. 8 c. 16 d Which of the following shows the correct order of steps for PCR? a. Melting DNA strands à Primer Binding à DNA polymerase replication b. Primer Binding à Melting DNA strands à DNA polymerase replication c. Melting DNA strands à DNA polymerase replication à Primer Binding Page 20

21 CONCEPT: DNA SEQUENCING DNA sequencing includes a variety of techniques to identify the nucleotide sequence of a molecule Dideoxy method (Sanger sequencing) was the first method of DNA sequencing in the 1970s - Dideoxy nucleotides (ddntps) were created without the 3 hydroxyl that facilitates nucleotide addition 1. Perform DNA amplification (PCR or other method) with normal nucleotides and low amount of ddntps 2. Addition of a ddntp would prevent further elongation of a sequence 3. Run DNA on a gel, which would show many different sized fragments - The different sizes were also colored, as the ddntp usually contains a dye - By reading the sizes and colors, you can determine the sequence of the molecule Today, more advanced technology is used to preform sequencing, which is must more accurate and fast EXAMPLE: ddttp ddatp ddgtp ddctp Sequence: TAGCCGCA Page 21

22 PRACTICE: 1. ddntps are unique because they do what? a. Speed up DNA replication b. Prevent the addition of more nucleotides c. Provide florescence to a DNA strand Page 22

23 CONCEPT: DNA LIBRARIES DNA libraries are collections of DNA A genomic library is a collection of genomic DNA fragments - Cut an organisms genome with restriction enzymes into many fragments - Put these fragments into yeast artificial chromosomes which can accommodate large DNA fragments - Up to 1 million base pairs - Then you can sequence these fragments, express them in bacteria or other organisms, etc EXAMPLE: Extract DNA Digest with Restriction Enzymes Put into Cloning vectors Express in Bacteria A cdna library is a collection of DNA fragments that represent the mrna transcribed in the cell - Start by isolating of an organism (represents the genes being expressed) - Reverse transcribe into DNA (all exons are gone) - Put this DNA into bacteria - Sequence to determine what genes are being expressed (constantly changing under different conditions) Page 23

24 EXAMPLE: Extract RNA Reverse Transcribe Into DNA Put into Cloning vectors Express in Bacteria PRACTICE: 1. Which of the following libraries consists of DNA that represents the mrna in the cell? a. Genomic library b. cdna library c. RNA library d. Protein library Page 24

25 CONCEPT: DNA TRANSFER INTO CELLS Often, scientists need to be able to transfer DNA cells Transfection is the process of transferring DNA molecules into cultured cells - Process can use chemicals, electricity (electroporation), or microinjection Transduction is viral-mediate gene transfer of DNA into cells - Use retrovirus that has been genetically engineered to contain the DNA of interest - Infection of the virus causes the DNA to enter the cell - Stable transformation occurs when the DNA is integrated into the host cell genome Transgenic organisms are organisms that have been genetically altered in some way - Includes addition of transgenes (foreign or modified genes) - Includes knockouts, which is when a gene has been inactivated or deleted EXAMPLE: Transduction and stable transformations Page 25

26 PRACTICE: 1. Which of the following methods uses electricity to get DNA into cells? a. Transfection b. Transduction c. Stable Transformation d. Transgenic Organism Creation 2. Which of the following methods can allow for foreign DNA to integrate into the host cell genome and remain there for extended periods of time? a. Transfection b. Transduction Page 26

27 CONCEPT: TRACKING PROTEINS IN CELLS It is important for researchers to be able to find and monitor a molecule s in a cell In situ hybridization can identify where a particular RNA or DNA is in the cell - Incubates probes labeled with fluorescence or radioactivity with fixed cells Reporter genes can be fused to proteins to identify a protein s location or movement in a cell - Green fluorescent protein (GFP) is a protein that has a green fluorescence - When fused to a recombinant protein, GFP will mark the protein of interest with green fluorescence When available, antibodies can also be used to identify a protein s location in a cell EXAMPLE: Page 27

28 CONCEPT: RNA INTERFERENCE RNA interference (RNAi) is a technique that can inactivate a gene RNAi uses non-coding RNAs like mirna, sirnas, or shrnas - These are into a cell or organisms - They are complementary to a DNA sequence and bind to that sequence - Act to specifically degrade or block expression of a gene inhibiting its function By inhibiting the gene, this allows scientists to determine the genes function - Determines phenotype in gene s absence - Allows for the gene to be replaced (recovery) EXAMPLE: Page 28

29 CONCEPT: GENETIC SCREENS Genetic screens allow researchers to evaluate the of thousands of genes at one time 1. Expose the organism to a mutagen in order to cause a lot of random mutations in the DNA - Many of these mutations will cause a phenotype in the organism 2. Search through all the organisms to find a few with a phenotype you want 3. Do further tests to identify the gene mutant causing the phenotype - This only works if the mutation is not lethal - Conditional mutants must be used to study lethal mutations - These mutations are only expressed under certain conditions (ex: temperature) EXAMPLE: Mutagen x Identify the gene Additional tests can be performed to identify the genes Complementation tests determines if a phenotype seen in 2+ organisms is due to a mutation in the same gene - Mate two homozygous recessive organisms - If phenotype is seen in the offspring then the mutation is in the same gene - If the phenotype is not seen in the offspring then the mutation is in different genes Epistasis analysis evaluates the order of protein pathways - If protein A acts before protein B (AàB) then: - A mutation in A will also stop the function of B - but a mutation in B will only stop the function of B and not A Page 29

30 EXAMPLE: Epistasis analysis A B X A B Blocks function of A and B A XB Blocks functions of only B PRACTICE: 1. Which test can be used to identify if two similar phenotypes in two different organisms are caused by mutations in the same gene? a. Genetic Screen b. Complementation Test c. Epistasis Analysis Page 30

31 2. What type of mutant is necessary to study lethal mutants? a. Non-lethal mutation b. Conditional Mutation c. Sensitive Mutation d. Epistatic Mutation Page 31

32 CONCEPT: BIOINFORMATICS Bioinformatics is the combination of computer science and biology to evaluate vast amounts of data DNA sequencing generates a lot of and bioinformatics helps organize it - Basic Local Alignment Tool (BLAST) is a software that can compare DNA or protein sequences - Examine conservation of genes among organisms, or variation within the human population Transcriptomics is the entire set of RNA molecules produced by a genome at any time Proteomes are the entire set of structural and functional properties of every protein produced by a genome Page 32