UNIT 4 Techniques used in Molecular Biology
Objectives On completion of this unit students will be able to: n n n Outline the steps in Polymerase Chain Reaction Analyse DNA agarose gel electrophoretograms Describe DNA hybridization and its application in probe synthesis Differentiate between Northern, Southern and Western Blots Outline the methods of DNA sequencing
Polymerase Chain Reaction n n The PCR technique is basically a primer extension reaction for amplifying specific nucleic acids in vitro. PCR will allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc. The particular stretch of DNA to be amplified is called the target sequence The target sequence is identified by a specific pair of DNA primers (oligonucleotides) usually about 20 nucleotides in length.
http: //stratfeed. cra. wallonie. be/img/page/PCR_web_page 5. jpg
Polymerase Chain Reaction n Primers must be duplicates of nucleotide sequences on either side of the piece of DNA of interest. n The exact order of the primers' nucleotides must already be known. n Primers can be constructed in the lab, or purchased from commercial suppliers.
Polymerase Chain Reaction The cycling reactions : n There are three major steps in a PCR, which are repeated for 30 or 40 cycles. n This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
Thermocycler
Steps in PCR
Three steps in PCR
Polymerase Chain Reaction n Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. n Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on.
Denaturation n 94°C n During the denaturation, the double strand melts open to single stranded DNA. n All enzymatic reactions stop (for example : the extension from a previous cycle).
Annealing/ Hybridization 54°C : hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. n The more stable bonds last a little bit longer (primers that fit exactly). n The polymerase can attach to pieces of double stranded DNA (template and primer), and starts copying the template. n Once there a few bases built in, the hydrogen bond is so strong between the template and the primer, that it does not break anymore.
Extension /Elongation 72°C : n This is the ideal working temperature for the polymerase. n Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment. n The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds d. NTP's from 5' to 3').
Polymerase Chain Reaction The use of a thermostable polymerase allows: n The dissociation of newly formed complimentary DNA n Subsequent annealing or hybridization of primers to the target sequence with minimal loss of enzymatic activity.
Polymerase Chain Reaction Is there a gene copied during PCR and is it the right size? n Before the PCR product is used in further applications, it has to be checked if there is a product formed. Factors that affect yield: n n n quality of the DNA is poor one of the primers doesn't fit too much starting template.
Polymerase Chain Reaction The product is of the right size: n It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. Factors that affect specificity: n one of the primers probably fits on a part of the gene closer to the other primer. n It is also possible that both primers fit on a totally different gene.
Uses of PCR 1. The method is especially useful for searching out disease organisms that are difficult or impossible to culture: n such as many kinds of bacteria, fungi, and viruses, because it can generate analyzable quantities of the organism's genetic material for identification.
Uses of PCR 2. PCR can also be more accurate than standard tests. n The technique is used to detect bacterial infections by detecting their DNA n n bacterial ear infection. Sensitive even when culture methods failed to detect it. lyme disease, the painful joint inflammation caused by bacteria transmitted through tick bites. , is usually diagnosed on the basis of symptom patterns. PCR can be used to identify the organism's DNA permitting speedy treatment that can prevent serious complications. PCR is the most sensitive and specific test for Helicobacter pylori, the disease organism now known to cause almost all stomach ulcers. It can detect the AIDS virus sooner during the first few weeks after infection than the standard ELISA test. PCR looks directly for the virus‘ unique nucleic acid, instead of the method employed by the standard test, which looks for indirect evidence that the virus is present by searching for antibodies the body has made against it. .
Uses of PCR 3. The method is also leading to new kinds of genetic testing. These tests diagnose not only people with inherited disorders, but also people who carry deleterious mutations that could be passed to their children.
Polymerase Chain Reaction 4. PCR can provide enormous peace of mind to people who are trying to have childrenn for example, by reassuring anxious parents-to-be that they run no risk of having a child with a particular genetic disease. n The technique even saves the lives of babies before they are born: detect whether the blood groups of mother and fetus are incompatible. This condition often leads to severe disability and even death of the fetus, but can be treated successfully in the womb with enough advance warning-thanks to PCR.
Polymerase Chain Reaction Animation PCR n http: //www. dnalc. org/ddnalc/resources/pcr. ht ml
Gel electrophoresis n a method that separates macromolecules-either nucleic acids or proteins-on the basis of: n size n electric charge n other physical properties, such as topology.
Pouring a gel
Loading a gel
Loading a gel
http: //elchem. kaist. ac. kr/vt/chem-ed/sep/electrop/graphics/eleczone. gif
https: //sites. google. com/a/luther. edu/genetics/_/rsrc/1235705828602/st udents/ashley-dissmore/protocolgel/gel%20 box. JPG? height=420&width=325
Gel electrophoresis n A gel is a colloid in a solid form. n The term electrophoresis describes the migration of charged particle under the influence of an electric field. n Electro refers to the energy of electricity. n Phoresis, from the Greek verb phoros, means "to carry across. "
Gel Electrophoresis n Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. n Activated electrodes at either end of the gel provide the driving force. n A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium.
Gel Electrophoresis n Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionisable groups. n These molecules exist in solution as electrically charged species either as cations (+) or anions (-) at a given p. H. n The charged particles will migrate either to the cathode or to the anode depending on the nature of their net charge.
Gel Electrophoresis n n DNA, is mixed in a buffer solution and applied to a gel. The electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a "molecular sieve, " separating the molecules by size. During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied.
Gel Electrophoresis The rate of migration through the electric field depends on: n The strength of the field n The size and shape of the molecules n The ionic strength and temperature of the buffer in which the molecules are moving.
Visualization n After staining, the separated macromolecules in each lane can be seen as a series of bands spread from one end of the gel to the other. n The ladder is a mixture of fragments with known size to compare with the unknown fragments.
Gel after staining
Gel Electrophoresis n Animation n http: //207. 4. 198/pub/flash/4/4. html n `http: //www. dnalc. org/ddnalc/resources/electroph oresis. html
Southern blotting n Southern blotting was named after Edward M. Southern who developed this procedure. n To oversimplify, DNA molecules are transferred from an agarose onto a membrane. n Southern blotting is designed to locate a particular sequence of DNA within a complex mixture.
Southern blotting n For example, Southern Blotting could be used to locate a particular gene within an entire genome. n The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. n Under optimal conditions, you can expect to detect 0. 1 pg of the DNA for which you are probing.
Southern blotting Steps in Southern blotting: n Digest the DNA with an appropriate restriction enzyme n Run the digest on an agarose n Denature the DNA (usually while it is still on the gel). For example, soak it in about 0. 5 M Na. OH. n Only ss. DNA can transfer.
Southern blotting n fragments greater than 15 kb are hard to transfer to the blotting membrane. n Depurination with HCl (about 0. 2 M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. n However, that the procedure may also be hampered by fragments that are too small.
Southern blotting n Transfer the denatured DNA to the membrane. n Traditionally, a nitrocellulose membrane is used, although nylon membrane may be used. Many scientists feel nylon is better since it binds more and is less fragile. n Transfer is usually done by capillary action, which takes several hours or using a vacuum blot apparatus which is faster).
Southern blotting n Capillary action transfer draws the buffer up by capillary action through the gel into the membrane, which will bind ss. DNA. n After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. n (You can also bake nitrocellulose at about 80 C for a couple of hours, but be aware that it is very combustible. )
Southern blotting n Probe the membrane with labeled ss. DNA. This is also known as hybridization. n This process relies on the ss. DNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.
Southern blot
Probe Detection n Visualize your labeled target sequence. n Probing is often done with 32 P labeled ATP, biotin/streptavidin or a bioluminescent probe. n If you used a radiolabeled 32 P probe, then you would visualize by autoradiograph. n Biotin/streptavidin detection is done by colorimetric methods. n Bioluminescent visualization uses luminesence.
Radioactive Detection
Probe Detection using Biotin/steptavidin n streptavidin is added which is an intermediary compound that will bind to the biotin on the probe. n Attached to the biotin is an enzyme such as alkaline phosphatase n A chromogenic substrate for the enzyme is added eg. BCIP/NBT (for alkaline phosphatase), which produces a blue-purple precipitate. n Therefore, visualization does not require X-ray film or other specific equipment.
Biotin/Streptavidin detection http: //www. fermentas. com/catalog/kits/img/chromogdet. gif
Biotin/Streptavidin detection http: //www. invitrogen. com/etc/medialib/en/images/ics_organized/brands/m olecular-probes. Par. 56035. Image. -1. 0. 1. gif
Northern Blotting n used for locating a sequence of RNA. n It is also known as Northern hybridization or RNA hybridization. n The procedure for and theory behind Northern blotting is almost identical to that of Southern blotting, except you are working with RNA instead of DNA.
Western Blotting n Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. n This method is, however, dependent on the use of a high -quality antibody directed against a desired protein.
Western Blotting n So you must be able to produce at least a small portion of the protein from a cloned DNA fragment to generate an antibody. n You will use this antibody as a probe to detect the protein of interest. n Western blotting tells you how much protein has accumulated in cells.
Steps in Western Blotting n Separate the proteins using SDS-polyacrylamide gel electrophoresis (also known as SDS-PAGE. This separates the proteins by size. n Place a nitrocellulose membrane on the gel and, using electrophoresis, drive the protein (polypeptide) bands onto the nitrocellulose membrane. n You want the negative charge to be on the side of the gel and the positive charge to be on the side of the nitrocellulose membrane to drive the negatively charged proteins over to the positively charged nitrocellulose membrane.
Steps in Western Blotting n This gives you a nitrocellulose membrane that is imprinted with the same protein bands as the gel. n Incubate the nitrocellulose membrane with a primary antibody. n The primary antibody, which is the specific antibody mentioned above, sticks to your protein and forms an antibody-protein complex with the protein of interest.
Steps in Western Blotting n Incubate the nitrocellulose membrane with a secondary antibody. n This antibody should be an antibody-enzyme conjugate. n The secondary antibody should be an antibody against the primary antibody. n This means the secondary antibody will "stick" to the primary antibody, just like the primary antibody "stuck" to the protein.
Steps in Western Blotting n The conjugated enzyme is there to allow you to visualize all of this. n To actually see your enzyme in action, you'll need to incubate it in a reaction mix that is specific for your enzyme. n You will see bands wherever there is a protein-primary antibodysecondary antibody-enzyme complex, or, in other words, wherever your protein is. n Put x-ray film on your membrane to detect a flash of light, which is given off by the enzyme or observe the color change produced by the enzyme with the substrate.
Western blot procedure http: //www. genscript. com/images/L 00204 -1. jpg
SDS Gel on left and Western blot on right http: //www. viswagenbiotech. com/images/sds_western_blot_proprep. jpg
Steps in making a primary antibody n Run the purified protein on an SDS-PAGE gel. n Stain the gel with KCl. n The KCl forms a precipitate with the SDS. n Since the area with the protein has a low concentration of SDS, the area with the protein will not show a precipitate. n This will allow you to see the protein band as a clear band against a milky white precipitate on the rest of the gel.
Steps in making a primary antibody n Carefully cut out the band, crush it and make an emulsion with 1 ml Freund's Complete Adjuvant (which is an oily substance). n The complete adjuvant contains bacteria (an immune stimulant) to increase the immune response. n Inject this subscapularly into a rabbit. n This is your first inoculation. Only use the complete adjuvant for the first inoculation.
Steps in making a primary antibody n Rest the rabbit for one month, repeat the process using an incomplete adjuvant. You can expect to see good antibody titers about 10 days after the second booster. n Bleed the rabbit. Now you have rabbit antisera. n To get your primary antibody, dilute the rabbit antisera in blotto (aka Carnation Nonfat Dry Instant Milk) and apply it to your nitrocellulose blot. Make sure you dilute 1: 500 to 1: 100 in blotto.
Steps in making a primary antibody http: //www. blogcdn. com/www. thecancerblog. com/media/2007/01/rat 2. jpg
Steps in making secondary antibody n This is much easier than the procedure for the primary antibody. n Grab a catalogue and look for a goat-anti-rabbit antibody conjugated to horseradish peroxidase (HRP). n The goat-anti-rabbit is your secondary antibody (the one that "sticks" to the primary antibody) and the HRP is the conjugated enzyme that will allow you to visualize your protein. n
DNA Sequencing determination of the precise sequence of nucleotides in a sample of DNA n first devised in 1975, n has become a powerful technique in molecular biology n allows analysis of genes at the nucleotide level. n has been applied to many areas of research. n
DNA Sequencing Two methods: n Enzymatic sequencing/chain termination (or 'Sanger-Coulson-Sequencing') n n The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains with these chains terminating at specific nucleotide positions. Chemical sequencing method ('Maxam-Gilbert. Sequencing’ Sequencing) n The sequence of a double-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions.
DNA Sequencing Both methods were equally popular to begin with n However the chain termination procedure is currently preferred, particularly for genome sequencing. n because the chemicals used in the chemical degradation method are toxic and therefore hazardous to the health of the researcher n because it has been easier to automate chain termination sequencing. n
Chain termination DNA sequencing based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis n The discovery of thermostable DNA polymerases, which led to the development of PCR has also resulted in new methodologies for chain termination sequencing n
Chain termination DNA sequencing n Thermal cycle sequencing has two advantages over traditional chain termination sequencing: It uses double-stranded rather than single-stranded DNA as the starting material. n Very little template DNA is needed, so the DNA does not have to be cloned before being sequenced. n
Chain termination DNA sequencing n Thermal cycle sequencing is carried out in a similar way to PCR but : n n n just one primer is used each reaction mixture includes one of the dd. NTPs Because there is only one primer, only one of the strands of the starting molecule is copied the product accumulates in a linear fashion, not exponentially The presence of the dd. NTP in the reaction mixture causes chain termination, the family of resulting strands can be analyzed and the sequence read by polyacrylamide gel electrophoresis
Chain termination sequencing The strand synthesis reaction: n is catalyzed by a DNA polymerase enzyme n requires the four d. NTPs (d. ATP, d. CTP, d. GTP , d. TTP) n would normally continue until several thousand nucleotides had been polymerized. n However, this does not occur in a chain termination sequencing experiment because: n n n as well as the four d. NTPs, a small amount of a dideoxynucleotide (e. g. dd. ATP) is added to the reaction. The polymerase enzyme does not discriminate between d. NTPs and dd. NTPs, so the dideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation because it lacks the 3′-hydroxyl group needed to form a connection with the next nucleotide. The result is therefore a set of new chains, all of different lengths
Chain termination sequencing Fluorolabeling has been important in the development of sequencing methodology, n because the detection system for fluorolabels has opened the way to automated sequence reading. n The label is attached to the dd. NTPs, with a different fluorolabel used for each one. n
Chain termination sequencing
Chain termination sequencing n Chains terminated with A are therefore labeled with one fluorophore (pink), n chains terminated with C are labeled with a second fluorophore (blue), n chains terminated with G are labeled with another fluorophore (yellow) n and chains terminated with T are labeled with another fluorophore (green).
Chain termination sequencing n Therefore, it is possible to : n n carry out the four sequencing reactions - for A, C, G and T - in a single tube load all four families of molecules into just one lane of the polyacrylamide gel, because the fluorescent detector can discriminate between the different labels and hence determine if each band represents an A, C, G or T. The sequence can be read directly as the bands pass in front of the detector and either printed out in a form readable by eye or sent straight to a computer for storage. When combined with robotic devices that prepare the sequencing reactions and load the gel, the fluorescent detection system provides a major increase in throughput and avoids errors that might arise when a sequence is read by eye and then entered manually into a computer.
Automated DNA Sequencer
Chain Termination sequencing http: //users. rcn. com/jkimball. ma. ultranet/B iology. Pages/F/Fluor. Dideoxy. Seq. gif
Sequence chromatogram
Animation of DNA sequencing n http: //www. dnalc. org/resources/animations/cy cseq. html
References http: //users. ugent. be/~avierstr/principles/pcrs teps. gif n http: //www. flmnh. ufl. edu/cowries/PCR. gif n
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