UNIT 4

1. UNIT 4 Techniques used in Molecular Biology

2. Objectives On completion of this unit students will be able to: • 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

3. Polymerase Chain Reaction • 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.

4. http://stratfeed.cra.wallonie.be/img/page/PCR_web_page5.jpg

5. Polymerase Chain Reaction • Primers must be duplicates of nucleotide sequences on either side of the piece of DNA of interest. • The exact order of the primers' nucleotides must already be known. • Primers can be constructed in the lab, or purchased from commercial suppliers.

6. Polymerase Chain Reaction The cycling reactions : • There are three major steps in a PCR, which are repeated for 30 or 40 cycles. • This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

7. Thermocycler

8. Steps in PCR

9. Three steps in PCR

10. Polymerase Chain Reaction • Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. • 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.

11. Denaturation • 94°C • During the denaturation, the double strand melts open to single stranded DNA. • All enzymatic reactions stop (for example : the extension from a previous cycle).

12. Annealing/ Hybridization 54°C :hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. • The more stable bonds last a little bit longer (primers that fit exactly). • The polymerase can attach to pieces of double stranded DNA (template and primer), and starts copying the template. • Once there are a few bases built in, the hydrogen bond is so strong between the template and the primer, that it does not break anymore.

13. Extension /Elongation 72°C : • This is the ideal working temperature for the polymerase. • 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. • The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3').

14. Polymerase Chain Reaction The use of a thermostable polymerase allows: • The dissociation of newly formed complimentary DNA • Subsequent annealing or hybridization of primers to the target sequence with minimal loss of enzymatic activity.

15. Polymerase Chain Reaction Is there a gene copied during PCR and is it the right size? • Before the PCR product is used in further applications, it has to be checked if there is a product formed. Factors that affect yield: • quality of the DNA is poor • one of the primers doesn't fit • too much starting template.

16. Polymerase Chain Reaction The product is of the right size: • 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: • one of the primers probably fits on a part of the gene closer to the other primer. • It is also possible that both primers fit on a totally different gene.

17. Uses of PCR 1. The method is especially useful for searching out disease organisms that are difficult or impossible to culture: • such as many kinds of bacteria, fungi, and viruses, because it can generate analyzable quantities of the organism's genetic material for identification.

18. Uses of PCR 2. PCR can also be more accurate than standard tests. • The technique is used to detect bacterial infections by detecting their DNA • 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..

19. 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.

20. Polymerase Chain Reaction 4. PCR can provide enormous peace of mind to people who are trying to have children- • for example, by reassuring anxious parents-to-be that they run no risk of having a child with a particular genetic disease. • 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.

21. Polymerase Chain Reaction Animation PCR • http://www.dnalc.org/ddnalc/resources/pcr.html

22. Gel electrophoresis • a method that separates macromolecules-either nucleic acids or proteins-on the basis of: • size • electric charge • other physical properties, such as topology.

23. Pouring a gel

24. Loading a gel

25. Loading a gel

26. http://elchem.kaist.ac.kr/vt/chem-ed/sep/electrop/graphics/eleczone.gifhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/electrop/graphics/eleczone.gif

27. https://sites.google.com/a/luther.edu/genetics/_/rsrc/1235705828602/students/ashley-dissmore/protocol-gel/gel%20box.JPG?height=420&width=325https://sites.google.com/a/luther.edu/genetics/_/rsrc/1235705828602/students/ashley-dissmore/protocol-gel/gel%20box.JPG?height=420&width=325

28. Gel electrophoresis • A gel is a colloid in a solid form. • The term electrophoresis describes the migration of charged particle under the influence of an electric field. • Electro refers to the energy of electricity. • Phoresis, from the Greek verb phoros, means "to carry across."

29. Gel Electrophoresis • Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. • Activated electrodes at either end of the gel provide the driving force. • A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

30. Gel Electrophoresis • Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, possess ionisable groups. • These molecules exist in solution as electrically charged species either as cations (+) or anions (-) at a given pH. • The charged particles will migrate either to the cathode or to the anode depending on the nature of their net charge.

31. Gel Electrophoresis • 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.

32. Gel Electrophoresis The rate of migration through the electric field depends on: • The strength of the field • The size and shape of the molecules • The ionic strength and temperature of the buffer in which the molecules are moving.

33. Visualization • 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. • The ladder is a mixture of fragments with known size to compare with the unknown fragments.

34. Gel after staining

35. Gel Electrophoresis • Animation • http://207.207.4.198/pub/flash/4/4.html • `http://www.dnalc.org/ddnalc/resources/electrophoresis.html

36. Southern blotting • Southern blotting was named after Edward M. Southern who developed this procedure. • To oversimplify, DNA molecules are transferred from an agarose onto a membrane. • Southern blotting is designed to locate a particular sequence of DNA within a complex mixture.

37. Southern blotting • For example, Southern Blotting could be used to locate a particular gene within an entire genome. • 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. • Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

38. Southern blotting Steps in Southern blotting: • Digest the DNA with an appropriate restriction enzyme • Run the digest on an agarose • Denature the DNA (usually while it is still on the gel).For example, soak it in about 0.5M NaOH. • Only ssDNA can transfer.

39. Southern blotting • fragments greater than 15 kb are hard to transfer to the blotting membrane. • Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. • However, that the procedure may also be hampered by fragments that are too small.

40. Southern blotting • Transfer the denatured DNA to the membrane. • 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. • Transfer is usually done by capillary action, which takes several hours or using a vacuum blot apparatus which is faster).

41. Southern blotting • Capillary action transfer draws the buffer up by capillary action through the gel into the membrane, which will bind ssDNA. • After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. • (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)

43. Southern blotting • Probe the membrane with labeled ssDNA. This is also known as hybridization. • This process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands.

44. Southern blot

45. Probe Detection • Visualize your labeled target sequence. • Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe. • If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. • Biotin/streptavidin detection is done by colorimetric methods. • Bioluminescent visualization uses luminesence.

46. Radioactive Detection

47. Probe Detection using Biotin/steptavidin • streptavidin is added which is an intermediary compound that will bind to the biotin on the probe. • Attached to the biotin is an enzyme such as alkaline phosphatase • A chromogenic substrate for the enzyme is added eg. BCIP/NBT (for alkaline phosphatase), which produces a blue-purple precipitate. • Therefore, visualization does not require X-ray film or other specific equipment.

48. Biotin/Streptavidin detection http://www.fermentas.com/catalog/kits/img/chromogdet.gif

49. Biotin/Streptavidin detection http://www.invitrogen.com/etc/medialib/en/images/ics_organized/brands/molecular-probes.Par.56035.Image.-1.0.1.gif

50. Northern Blotting • used for locating a sequence of RNA. • It is also known as Northern hybridization or RNA hybridization. • 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.