Lecture 3 - Tan (1) - Radioactivity, Safety & Detection Methods PDF

Today’s Outline 1) Optical Spectroscopy • Absorption of UV and visible light • Fluorescence spectroscopy 2) Molecular Recognition • • • • Modes of detection Recognition of nucleic acids Recognition of proteins Biosensors 41 Modes of Detection • Many methods have been developed to probe for the p...

Today’s Outline 1) Optical Spectroscopy • Absorption of UV and visible light • Fluorescence spectroscopy 2) Molecular Recognition • • • • Modes of detection Recognition of nucleic acids Recognition of proteins Biosensors 41 Modes of Detection • Many methods have been developed to probe for the presence of certain DNA or RNA sequences or certain proteins. • Ideally, we should be able to “see” the final results, although DNA, RNA, and proteins are really far too small to be seen by the naked eye. • There are two modes of detection that are commonly used: (1) Fluorescence (2) Radioactivity 42 Radioactivity • A radioactive isotope (also called radioisotope) is any of several species of the same chemical element with different masses whose nuclei are unstable and dissipate excess energy by spontaneously emitting radiation. The isotopes have the same number of protons but different number of neutrons. • Radioisotopes are commonly used to detect very small amounts of DNA, RNA, or protein (in the femtogram [1x10-15 gram] to picogram [1x10-12 gram] levels). • A radioactive isotope is introduced into DNA, RNA, or protein for quantification purposes and its presence can be detected by: - sensitive radiation detectors such as Geiger counters and liquid scintillation counters - exposure to X-ray films (autoradiography) or phosphor storage screens 43 Common radioisotopes used 32P is frequently used for detecting DNA and RNA and has the highest emission energy of all common research radioisotopes. This is a major advantage in experiments for which sensitivity is a primary consideration. Its maximum specific activity is 9131 Ci/mmol. 35S is used to label proteins and nucleic acids. Cysteine is an amino acid containing a thiol group (-SH), which can be labelled by S-35. Since nucleotides do not contain a sulfur group, the oxygen on one of the phosphate groups can be substituted with a sulfur. This thiophosphate acts the same as a normal phosphate group, although there is a slight bias against it by most polymerases. The maximum theoretical specific activity is 1,494 Ci/mmol. 3H is used to detect DNA and RNA. It is a very low energy emitter, with a maximum theoretical specific activity of 28.8 Ci/mmol. However, there is often more than one tritium atom per molecule: for example, tritiated UTP is sold by most suppliers with carbons 5 and 6 each bonded to a tritium atom. 125I is used to radiolabel proteins, usually at tyrosine residues. Unbound iodine is volatile and must be handled in a fume hood. Its maximum specific activity is 44 2,176 Ci/mmol. How long do radioisotopes last? • The term half-life is defined as the time it takes for one-half of the atoms of a radioactive material to decay. It is independent of the original quantity. • Different radioisotopes have different half-lifes. For example, P-32 has a half-life of 14.29 days, S-35 has a half-life of 87 days, while C-14 has a long half-life of 5,730 years. 45 Radiation sickness Radiation sickness is a condition where there is damage to the body occurring as a result of large doses of radiation received by the body over a short period of time. The radiation causes cellular degradation due to damage to DNA and other key molecular structures within the cells. Eyes: High doses can trigger cataracts months later. Thyroid: Hormone glands vulnerable to cancer. Radioactive iodine builds up in thyroid. Children most at risk. Lungs: Vulnerable to DNA damage when radioactive material is breathed in. Stomach: Vulnerable if radioactive material is swallowed. Reproductive organs: High doses can cause sterility. Skin: High doses cause redness and burning. Bone marrow: Site of production of red and white blood cells. Radiation can lead 46 to leukemia and other immune system diseases. Safety in using radioactivity We need to be careful when using radioactivity in the lab. For example, the high-energy beta emissions from 32P can present a substantial skin and eye dose hazard. Common safety measures include the following: - Designate special area for handling radioisotopes and clearly label all containers. - Store radioisotopes like 32P behind lead shielding. - Work behind acrylic glass shields and wear safety goggles to protect eyes from radiation. - Practise routine operations to improve dexterity and speed before using radioisotopes like 32P. - Handle potentially volatile chemical forms in ventilated enclosures. - Isolate radioactive waste in clearly labelled shielded containers and hold for decay. - Always check work area and work clothes (e.g. lab coat) for accidental spills after every experiment. 47 - Go for regular health checks to detect symptoms of radiation sickness. Today’s Outline 1) Optical Spectroscopy • Absorption of UV and visible light • Fluorescence spectroscopy 2) Molecular Recognition • • • • Modes of detection Recognition of nucleic acids Recognition of proteins Biosensors 48 What are restriction endonucleases? Restriction endonucleases (or restriction enzymes) recognize and cleave specific DNA sequences, i.e. they are DNA-cutting enzymes that only cleave at particular positions. The sequence of nucleotides that is recognized by each restriction enzyme is known as its restriction site. For example, BamHI recognizes GGATCC, while EcoRI recognizes GAATTC. The enzymes can cut their DNA substrate within the recognition site or outside the recognition site (i.e. the recognition and cleavage sites may be separate from each other). 49 Where do restriction enzymes come from? • They are usually isolated from bacteria • Restriction enzymes are usually named after the bacteria they are isolated from. For example: EcoRI – isolated from E. coli strain R HindIII – isolated from Haemophilus influenzae strain Rd • Nobel Prize in 1978 was awarded for discovery of restriction enzymes 50 If bacteria produce these enzymes, why isn’t the bacterial DNA digested by them?  Probability of finding a target of: 4 bases – 1/256 5 bases – 1/1024 6 bases – 1/4096 8 bases – 1/65,536  Restriction endonucleases protect bacteria from invasion by foreign DNA (like phage) (Restrict the host range of the virus)  Bacteria use methylation to protect their chromosomal DNA; invading viral DNA is not methylated  Many restriction enzymes cannot cleave methylated DNA 51 Restriction enzymes often function as homodimers 5' G A A T T C EcoRI on DNA C T T A A G 5' a cartoon view Hence, restriction sites are often (but not always) palindromic. 52 Sticky ends vs. blunt ends Upon cleavage, restriction enzymes often leave a single stranded overhang (“sticky” end). The overhang can be either 5’ or 3’. Sometimes restriction enzymes leave a “blunt” end. (5’ overhang) 53 5’-TCAGATCGTACTTGAGAATTCGGGCT-3’ 3’-AGTCTAGCATGAACTCTTAAGCCCGA-5’ EcoRI 5’-TCAGATCGTACTTGAG 3’-AGTCTAGCATGAACTCTTAA AATTCGGGCT-3’ GCCCGA-5’ Sticky ends 54 5’-TCAGATCGTACTTGAGAATTCGGGCT-3’ Fragment 1 3’-AGTCTAGCATGAACTCTTAAGCCCGA-5’ Fragment 2 5’-CTAGGACCGAATTCAAGTACGGACC-3’ 3’-GATCCTGGCTTAAGTTCATGCCTGG-5’ EcoRI 5’-TCAGATCGTACTTGAG 3’-AGTCTAGCATGAACTCTTAA AATTCGGGCT-3’ GCCCGA-5’ 5’-CTAGGACCG AATTCAAGTACGGACC 3’ 3’-GATCCTGGCTTAA GTTCATGCCTGG 5’ 55 A new recombinant DNA molecule 5’-TCAGATCGTACTTGAGAATTCAAGTACGGACC-3’ 3’-AGTCTAGCATGAACTCTTAAGTTCATGCCTGG-5’ DNA ligase seals the nicks between the two strands, reforming covalent phosphodiester bonds 56 What is happening in the DNA backbone? Restriction Enzyme DNA Ligase 57 Analogies Restriction Enzyme DNA Ligase Summary of DNA cut-and-paste Examples of restriction enzymes EcoRI SmaI BamHI XmaI KpnI NotI Hundreds of enzymes are available (for example, see: http://www.neb.sg/products/restriction-endonucleases) 60 Type II vs type IIs restriction enzymes • Type II enzymes cut within their recognition sequences. EcoRI: BamHI: • Type IIs enzymes cut outside of their recognition sequences. BsmBI: BbsI: BsaI: The “N”s are useful for designing unique overhangs Overview of Southern Blot A Southern blot is a method for detection of a specific DNA sequence in DNA samples. It was invented by Sir Edwin Southern, Professor Emeritus at University of Oxford. 62 Details of Southern Blot Procedure 1) Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. 2) The DNA fragments are then electrophoresed on an agarose gel to separate them by size. 3) The DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA and destroy any residual RNA that may still be present. 4) A sheet of nitrocellulose (or nylon) membrane is placed on top of the gel. Pressure is applied evenly to the gel (by placing a stack of paper towels on top of the membrane and gel) to ensure good and even contact between gel and membrane. Buffer transfer by capillary action is then used to move the DNA from the gel onto the membrane. 5) The membrane is then baked at 80oC for 2 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane. 6) The membrane is then exposed to a hybridization probe. The probe is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and detergents such as SDS to reduce nonspecific binding. 7) After hybridization, excess probe is washed from the membrane and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of colour on the membrane if a chromogenic 63 detection method is used. Restriction fragment length polymorphism • Restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences. • The basic technique for the detection of RFLPs involves: - Fragmenting a sample of DNA using a restriction enzyme - Separating the resulting DNA fragments by length using agarose gel electrophoresis - Transferring the DNA to a membrane via Southern blot - Hybridizing to the membrane a labeled DNA probe to determine the length of the fragments that are complementary to the probe. • An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele. 64 Example 1 of RFLP A small segment of the genome is being detected by a DNA probe (thicker line). In allele "A", the genome is cleaved by a restriction enzyme at three nearby sites (triangles), but only the rightmost fragment will be detected by the probe. In allele "a", restriction site 2 has been lost by a mutation, so the probe now detects the larger fused fragment running from sites 1 to 3. The second diagram shows how this fragment size variation would look on a Southern blot, and how each allele (two per individual) might be inherited in members of a family. 65 Example 2 of RFLP The probe and restriction enzyme are chosen to detect a region of the genome that includes a variable number tandem repeat segment (boxes in schematic diagram). In allele "c" there are five repeats in the VNTR, and the probe detects a longer fragment between the two restriction sites. In allele "d" there are only two repeats in the VNTR, so the probe detects a shorter fragment between the same two restriction sites. Applicability to diseases: • Trinucleotide repeat disorders are a set of genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide repeats in certain genes exceed the normal, stable threshold (which varies from gene to gene). • Currently, nine neurologic disorders (e.g. Huntington’s disease) are known to be caused by an increased number of CAG repeats, typically in coding regions. During protein synthesis, the expanded CAG repeats are translated into a series of uninterrupted glutamine residues forming what is known as a polyglutamine tract ("polyQ"). Such polyglutamine tracts may be subject to increased aggregation. Some RNA-binding proteins may also recognize and bind to the CAG repeats to cause disease. 66 What is Northern Blot? • A variation of Southern blot, but used for the detection of RNA instead. • Developed in 1977 at Stanford University. • Totally cellular RNA is separated by size, transferred to a membrane (blotted), and detected by a complementary (radioactive-labelled) probe that hybridizes to a specific species of RNA. • Used in studies of gene expression, e.g. to determine whether a specific mRNA is present in different cell types. • Reveals information about the RNA identity, size, and abundance. • Only measures steady state RNA levels and not transcription rates or RNA stability. 67 Northern Blot Workflow • Similar to Southern blot • mRNA (RNAs with a poly(A) tail) can be isolated using oligo (dT) beads • RNAs have to be denatured, for example, using glyoxal 68 Fluorescence in situ hybridization • Fluorescence in situ hybridization (FISH) is originally a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. • It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. • FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA, and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression. 69 Example of DNA-FISH FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. A cell positive for the bcr-abl rearrangement (associated with chronic myelogenous leukemia) using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (upper left) is the one where the rearrangement is present. 70 Example of RNA-FISH Single-cell identification in microbial communities - Based on the patchy conservation of rRNA, 71 oligonucleotide probes can be designed with specificities to particular types of microorganisms. DNA arrays • In traditional biomolecular methods, one gene is analyzed at a time. This becomes very time consuming and tedious. • In recent years, a new technology has become available that allows massively parallel analysis on a single device, the so-called DNA chip or DNA microarray. • This technique makes use of the molecular recognition of two strands of oligonucleotides which only bind to each other (hybridise) if they are complementary to each other. 72 Principle of DNA Arrays On a DNA array, there are large numbers of DNA molecules or oligonucleotides which are immobilised onto a substrate like a glass slide or a nylon membrane in the form of spots. Labelled DNA samples can only hybridize with certain spots on the array, i.e. those containing a matching oligonucleotide sequence. This results in a characteristic pattern (a fingerprint) of colored and uncolored spots. 73 Cy3 and Cy5 are commonly used Two DNA samples that are differentially labeled can be hybridized to the same array. 74 Genes can be expressed with different efficiencies • Gene A is transcribed and translated much more efficiently than gene B. Transcriptome analysis - DNA microarrays Single-stranded DNA probes are adhered to the surface of the chip Bright red: up-regulation Bright green: down-regulation Yellow: no change Today’s Outline 1) Optical Spectroscopy • Absorption of UV and visible light • Fluorescence spectroscopy 2) Molecular Recognition • • • • Modes of detection Recognition of nucleic acids Recognition of proteins Biosensors 77 What are antibodies? An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to identify and neutralize pathogens such as bacteria and viruses. Each antibody consists of four subunits – two identical light chains (L), with a molecular weight of about 25kDa, and two identical heavy chains (H), with a molecular weight of about 50kDa. These subunits are associated via disulfide bonds and non-covalent interactions. There are five classes of antibodies: IgA, IgD, IgE, IgG (most common in the body), and IgM. These classes are determined by the five different types of heavy chains. There are also two types of light chains, which can appear in any of the five Ig classes. 78 More details on antibodies Within the antibody molecule, there are constant (C) and variable (V) regions. The constant regions are the same for every antibody of that class. The variable regions make up the paratopes of the antibody, which target and bind to the antigen of interest. The paratopes are located at the N-terminal tips of the Y-shape. Each antibody molecule has two identical paratopes for the antigen. Hence, the antibody is bivalent. There are two types of antibodies: (1) Polyclonal antibodies are a mixture of antibodies as produced by a host upon injection of an antigen. They can bind to several different parts (epitopes) on the antigen. (2) Monoclonal antibodies bind to only one particular epitope on the antigen. They are more specific and reproducible. 79 Cleavage of antibodies Immunoglobulins can be cleaved into fragments: (1) The enzyme papain cleaves IgG into three fragments – two identical Fab fragments originating from the arms of the Y-shape and a Fc fragment from the stem of the Y-shape. As the Fab fragments contain the paratopes, they retain the antigen binding (ab) ability. (2) The enzyme pepsin cleaves an antibody at the stem below the hinge region, resulting in a F(ab)2 fragment with the arms of the Y-shape still being joined. This F(ab)2 fragment contains both paratopes. Occasionally, the Fab or F(ab)2 fragments are used in immunoassays, instead of the whole immunoglobulin molecule. 80 Antigens An antigen is a molecule (e.g. a toxin or other foreign substance) that induces an immune response in the body, especially the production of antibodies. There are two classes of antigens: (1) Complete or full antigens induce an immune response by themselves. They are usually reasonably sized proteins (kDa range). These full antigens can have several copies of the same epitope or they can contain several different epitopes that bind to different antibodies (multi-determinant). (2) Incomplete antigens, also called haptens, are lower molecular weight molecules like the drug theophylline (180Da) or the steroid hormone cortisone (362Da). They cannot induce an immune response by themselves. However, if attached to a protein carrier, the production of specific antibodies against these haptens can be triggered. Once produced, these anti-hapten antibodies will recognize the hapten even without the protein carrier. Haptens usually only feature a single epitope. 81 Details on epitopes • An epitope (binding site of the antigen) makes up only a small area of the total antigen structure. • Epitopes can be: (1) Continuous – common in fibrous proteins (2) Discontinuous – common in globular proteins and helical structures and are generated through folding. Discontinuous epitopes can be destroyed upon denaturation, for example, if disulfide bonds are split. • Antibodies are capable of distinguishing between antigens even if they are chemically very similar. It is the overall three-dimensional structure of the antigen that defines its affinity and interaction with an antibody. 82 Antibody-antigen complex formation • If the paratope of the antibody matches the epitope of any antigen, an AbAg complex is formed. • An antibody only reacts with a matching antigen and with this specific antigen only. This high specificity enables direct analysis of complex sample mixtures, such as blood and urine. • The affinity of antibody to antigen is very high and binding occurs even at very low concentrations. Hence, immunoassays typically have high sensitivity (low limits of detection). 83 Competitive immunoassays • A limited amount of antibody is used, which is insufficient to bind with all the antigen molecules of the assay. • The sample containing an unknown amount of antigen is mixed with a fixed and known amount of labelled antigen. • The unlabelled and labelled antigens compete for binding to the antibody. 84 Non-competitive immunoassays • The antigen sample is incubated with an excess of antibody reagent, hence not all the antibody binding sites will be occupied. • To detect the amount of antigen attached to an antibody, a second labelled antibody is added, which binds to another epitope of the antigen. • Non-competitive assays are better suited for large analyte molecules, which are likely to have several epitopes (at least two binding sites are required on the analyte molecule). 85 Home Pregnancy Test • The concentration of the hormone human chorionic gonadotropin (hCG) (which appears in the urine) increases rapidly during the first weeks of pregnancy. • The adsorbent test strip is encased in plastic with a sample input window, a test window, and a (positive) control window. • A drop of urine is applied at the input window and the liquid moves along the strip by capillary action. • There are two types of antibodies on the strip that recognize hCG – (1) the capture antibody that is covalently fixed to the device surface; (2) the tracer antibody, which is labelled with a dye and is impregnated on the surface but not permanently attached. 86 Home Pregnancy Test • If hCG is present, it forms a complex with the tracer antibody as the urine flows along the test strip. • The complex then continues to move along the adsorbent material and passes over the area with the immobilized capture antibody. • A sandwich is formed, with hCG in between the immobilized capture antibody and the tracer antibody. • The amount of sandwich complexes formed is directly proportional to the amount of hCG present. If the hCG concentration exceeds a threshold, the dye color becomes visible to the naked eye. 87 Overview of ELISA In ELISA (enzyme-linked immunosorbent assay), the enzyme label acts as a catalyst for the conversion of a colourless substrate to a coloured product. One single enzyme molecule can catalyse the conversion of a large number of substrate molecules and thus generate a large signal. This signal amplification enables the quantitative analysis of low sample concentrations. For example, hormones in blood are often analysed with ELISA. 88 ELISA for HIV • HIV antigens are immobilized on the surface of a microtitre well. • The sample, possibly containing HIV antibodies, is added and then left to incubate. • After washing, a secondary antibody with an enzymatic label and targeted towards the HIV antibodies is added. This secondary antibody binds to the HIV antibodies, if they are present. • After another wash, the appropriate enzyme substrate is added. If any secondary antibodies are bound, then a colour reaction occurs. • The colour intensity can be measured with a spectrophotometer. 89 Western Blot Western Blot is a technique to detect specific proteins in a biological sample. It uses gel electrophoresis to separate proteins. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. The gel electrophoresis step is included in western blot analysis to resolve the issue of the cross-reactivity of antibodies. 90 Detection in Western Blot The secondary antibody is commonly linked to an enzyme called horseradish peroxidase (HRP). HRP catalyzes the conversion of chromogenic substrates into colored products, and produces light when acting on chemiluminescent substrates. In general, detection by chemiluminescent substrates is favoured. The sensitivity is 10- to 100-fold greater, and quantifying of light emission is possible over a wide dynamic range, whereas that for coloured precipitates is much more limited, about one order of magnitude less. Stripping filters are also much easier when chemiluminescent substrates are used. 91 Measuring protein stability The half-life of a protein can be determined by a pulse-chase experiment: Pulse of 35S is added to cells followed by a chase of cold methionine. At regular time points, cells are harvested and lysed. The protein-of-interest is immunoprecipitated using an antibody. Half-life, t1/2 = ln2 / , where  = degradation constant 92 Thermal Shift Assay 93 Today’s Outline 1) Optical Spectroscopy • Absorption of UV and visible light • Fluorescence spectroscopy 2) Molecular Recognition • • • • Modes of detection Recognition of nucleic acids Recognition of proteins Biosensors 94 What is a biosensor? A biosensor consists of a bioreceptor with a specific surface to detect a target analyte and a transducer to pass on a signal when recognition occurs, i.e. Biosensor = molecular recognition + signal transduction (in a single device) Devices can be used by non-specialist operators at the point-of-care and this allows for immediate action to be taken, if any. 95 Bioreceptor A bioreceptor must react specifically with an analyte of interest. Usually, the bioreceptor molecules are immobilized at, or close to, the surface of the transducer. Enzymes are the most common bioreceptors. Only small quantitites of enzyme molecules are required because they can regenerate themselves after reacting. Enzymes are also highly specific in their action. Antibodies are another type of biomolecule commonly used as bioreceptors. Antibodies are highly specific for their targets. However, unlike enzymes, they can only be used for a one-time measurement. After reaction, they have to be disposed or the antigens have to be washed off with suitable reagents. The bioreceptor does not have to be an enzyme or antibody. Virtually any compound that exhibits molecular recognition for an analyte is suitable. This could be nucleic acids (due to complementary base pairing) or even living cells. However, enzymes and antibodies are relatively easy to incorporate into a device. Biological cells must be supplied with nutrients in order to keep them alive. 96 Transducer A transducer is the component that responds to molecular recognition and converts this response to an output that can be amplified, stored, or displayed. The type of molecular recognition determines the form of the transducer used: (1) Enzymatic reactions often involve electron transfer. This electrical activity can be measured with amperometric, potentiometric, or conductometric sensors. (2) If the bioreaction involves the generation of H+ or OH-, then a pH sensitive dye in combination with an optical device can be used. (3) For antibody-antigen binding, the mass change on the surface of the transducer can be detected with a piezoelectric device. (4) Exothermic or endothermic reactions can be followed with a temperature sensor. 97 Glucose biosensor • The blood glucose sensor is the most successful commercial biosensor. • About 9% of the adult population in Singapore have diabetes. • The highly specific enzyme glucose oxidase (GOx) is employed as a bioreceptor. • GOx is a near ideal bioreceptor because: - It can be produced cheaply by soil fungi; - It withstands greater extremes of pH, ionic strength, and temperature than many other enzymes; - It reacts readily over the concentration range of glucose encountered in human blood samples; - The current produced is directly proportional to the amount of glucose in the sample. 98 Operating principle The tip of the sensor is made of a membrane selectively permeable to glucose. Once the glucose passes through the membrane, it is oxidized by glucose oxidase. Reduced glucose oxidase can then be regenerated (oxidized) by reacting with molecular oxygen, forming hydrogen peroxide as a by-product. At the electrode surface, hydrogen peroxide is oxidized into water, generating a current which can be measured and correlated with the glucose concentration outside the membrane. (Alternatively, ferrocenium/ ferrocene can be used in place of oxygen/ hydrogen peroxide.) 99