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- 1. Chapter 3 Recombinant DNA Technology and Genomics
- 2. Agarose Gel Electrophoresis Electrophoresis is a molecular technique that separates nucleic acids and proteins based on: Size and +-+Charge+-+
- 3. DNA is a negatively charged molecule and therefore is attracted to positive charges. Agarose Gel Electrophoresis
- 4. Agarose provides a matrix through which DNA molecules migrate. Larger molecules move through the matrix slower than small molecules The higher the concentration of agarose, the better the separation of smaller molecules Agarose Gel Electrophoresis
- 5. How to make an agarose gel: 1. Weigh out a specified amount of agarose powder. 2. Add the correct amount of buffer. 3. Dissolve the agarose by boiling the solution. 4. Pour the gel in a casting tray. 5. Wait for the gel to polymerize. Agarose Gel Electrophoresis
- 6. How to make an agarose gel: 6. Place gel in chamber and cover with buffer 7. Add loading dye to the sample 8. Load sample on to the gel. Agarose Gel Electrophoresis
- 7. How to make an agarose gel: 9. Stain the gel 10.Take a picture of the gel 11.Analyze results Agarose Gel Electrophoresis
- 8. Agarose Gel Electrophoresis Electrophoresis Animation
- 9. Recombinant DNA Recombinant DNA technology Allows DNA to be combined from different sources Also called genetic engineering or transgenics
- 10. Vector – DNA source which can replicate and is used to carry foreign genes or DNA fragments. Recombinant DNA – A vector that has taken up a foreign piece of DNA. Recombinant DNA
- 11. Restriction enzyme – an enzyme which binds to DNA at a specific base sequence and then cuts the DNA Restriction enzymes are named after the bacteria from which they were isolated. • Bacteria use restriction enzymes to chop up foreign viral DNA Restriction Enzymes
- 12. Recognition site – specific base sequence on DNA where a restriction enzyme binds. • All recognition sites are palindromes, which means they read the same way forward and backward. example: RACECAR or GAATTC CTTAAG • Each restriction enzyme has its own unique recognition site. Restriction Enzymes
- 14. After cutting DNA with restriction enzymes, the fragments can be separated on an agarose gel. • The smaller fragments will migrate further than the longer fragments in an electric field. • The bands are compared to standard DNA of known sizes. This is often called a DNA marker, or a DNA ladder. Restriction Enzymes
- 15. Restriction Enzymes Running a Restriction digest on an agarose gel
- 16. Restriction Enzymes After analyzing your results, you draw a restriction map of the cut sites. • A restriction map is a diagram of DNA showing the cut sites of a series of restriction enzymes.
- 19. Most restriction enzymes cut within the recognition site. When restriction enzymes cut in a zig zag pattern, sticky ends are generated. Restriction Enzymes
- 20. Overhanging sticky ends will complementarily base pair, creating a recombinant DNA molecule. DNA ligase will seal the nick in the phosphodiester backbone. Restriction Enzymes
- 21. Restriction Enzymes Restriction Enzyme Animation
- 22. Transformation Transformation – the process by which organisms take up and express foreign DNA Griffith’s experiment
- 23. Bacterial Transformation Bacteria, such as E.coli, can take up and express foreign DNA, usually in the form of a plasmid. Transformation
- 24. Gene cloning – using bacterial transformation to make lots of copies of a desired gene. Transformation Gene Cloning Animation
- 25. Transformation Steps of Bacterial Transformation 1. Choose a bacterial host a. E. coli is a model organism i. Well studied ii. No nuclear membranes iii. Has enzymes necessary for replication iv. Grows rapidly (20 min. generation time) v. Inexpensive vi. Normally not pathogenic vii. Easy to work with and transform
- 26. Transformation Steps of Bacterial Transformation 2. Choose a plasmid to transform a. Characteristics of a useful plasmid i. Single recognition site • Plasmid only cuts in one place, so this ensures that the plasmid is reformed in the correct order. ii. Origin of replication • Allows plasmid to replicate and make copies for new cells. iii. Marker genes • Identifies cells that have been transformed. gene for antibiotic resistance – bacteria is plated on media with an antibiotic, and only bacteria that have taken up a plasmid will grow gene that expresses color – bacteria that have taken up a recombinant plasmid are a different color than bacteria that have taken up a NONrecombinant vector.
- 27. Transformation Steps of Bacterial Transformation 3. Prepare bacterial cells for transformation a. Treat with calcium chloride – softens the phospholipid bilayer of the cell membrane, which allows the plasmid to pass through b. Electroporation – brief electric pulse c. Directly inject plasmid into bacterial host
- 28. Transformation Steps of Bacterial Transformation 4. Plate transformation on appropriate media a. Contains nutrients for bacteria and antibiotic to distinguish transformed bacteria from NONtransformed bacteria 5. Incubate plates overnight a. E.coli grows at body temp. (37 °C) 6. Analyze plates Gene Cloning Animation
- 29. Gene Cloning What makes a good vector?
- 30. Gene Cloning What makes a good vector?
- 31. Gene Cloning How do you identify and clone a gene of interest? • BUILD A LIBRARY!! • DNA library – a collection of cloned DNA fragments from a particular organism • Can be saved for a relatively long period of time and screened to pick out different genes of interest • Two types of libraries 1. Genomic library – contains DNA sequences from entire genome 2. cDNA library – contains DNA copies of mRNA molecules expressed Construction of a DNA library Animation
- 32. Gene Cloning
- 33. Gene Cloning Steps to screen a library 1. Plate cells and transfer to nylon membrane 2. Lyse bacterial cells 3. Denature DNA 4. Add radioactively labeled probe that is complementary to gene of interest
- 34. Gene Cloning Steps to screen a library 5. Wash off non-specifically bound probe 6. Expose membrane to x-ray film 7. Align exposed film with original plate 8. Grow cells containing gene of interest in culture.
- 35. Gene Cloning Rarely is an entire gene cloned in one piece, even in a cDNA library, therefore must “walk” the chromosome until a start and stop codon are found.
- 36. Sequencing Sequencing – determining the order and arrangement of G’s, A’s, T’s and C’s in a segment of DNA.
- 38. The Sanger sequencing method uses dideoxy- nucleotides to generate all possible fragments of the DNA molecule to be sequenced. Sequencing deoxynucleotide dideoxynucleotide
- 39. Set up four different reactions: Sequencing
- 40. Load the four reactions in different wells of a polyacrylamide gel to separate the fragments Sequencing
- 42. Human Genome Project Initiated in 1990 with plan to identify all human genes • Analyze genetic variation among humans • Map and sequence genomes of model organisms • Develop new lab technology • Disseminate genome information • Consider ethical, legal, and social issues that accompany genetic research
- 43. Human Genome Project Francis Collins Craig Venter
- 44. Human Genome Project Consider ethical, legal and social issues • Who owns your DNA?
- 45. Develop new lab technology • Automated Sequencing Human Genome Project
- 46. Human Genome Project Disseminate genome information • GenBank database
- 47. Human Genome Project Analyze genetic variation among humans • The genome is approximately 99.9% identical between individuals of all nationalities and backgrounds.
- 48. Human Genome Project Map and sequence genomes of model organisms • E.coli • Arabidopsis thaliana • Saccharomyces cerevisiae • Drosophila melangaster • Caenorhabditis elegans • mus musculus
- 49. PCR Polymerase chain reaction (PCR) A lab technique used to amplify segments of DNA "PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate and reproduce DNA. It makes abundant what was once scarce -- the genetic material required for experimentations."
- 50. PCR Reaction requirements Template DNA – total genomic DNA isolated from an organism that contains a target region to be amplified DNA primers - Short pieces of single stranded DNA that flank the target Taq DNA polymerase - Attaches nucleotides on the growing strand of DNA Nucleotides (GATC) – Polymerase adds complementary nucleotides to the template
- 51. PCR Reactions are placed in a machine called a thermal cycler. The machine cycles through three temperatures.
- 52. PCR 1. Heat samples to 94°C for a minute or so to denature the double stranded template DNA.
- 53. PCR 2. Drop temperature to around 50 or 60°C to allow primers to anneal.
- 54. PCR 3. Maintain temperature at 72°C for a minute or two to allow the polymerase to elongate the new DNA strands.
- 55. PCR The thermal cycler repeats the denaturing, annealing, and elongating temperatures approximately 30 times. PCR Animation
- 56. PCR PCR amplification is logarithmic, meaning the number of copies of the target is doubled every cycle. (2n)
- 58. PCR Cloning by PCR • Design primer specific for gene of interest (must know some of the sequence) • Can use a T-vector because Taq polymerase adds an A to the 3’ end of sequence
- 59. Applications of Recombinant DNA Technology
- 60. Chromosomal Location and Gene Copy Number Fluorescence in situ hybridization (FISH)
- 61. Southern Blot - molecular technique where DNA is transferred onto a membrane from an agarose gel and a probe is hybridized. Chromosomal Location and Gene Copy Number
- 62. Southern Blot The first step in preparing a Southern Blot is to cut genomic DNA and run on an agarose gel.
- 63. Southern Blot The next step is to blot or transfer single stranded DNA fragments on to a nylon membrane.
- 64. The next step is to hybridize a radioactively labeled DNA probe to specific sequences on the membrane. Southern Blot
- 65. Southern Blot The last step is to expose the radioactively labeled membrane to a large sheet of film. You will only visualize bands where the probe hybridized to the DNA..
- 66. Southern Blot Animation Southern Blot
- 67. Studying Gene Expression Northern Blot • Isolate RNA from tissue of interest • Separate on agarose gel • Blot onto nylon membrane • Hybridize probe specific for desired transcript • Expose on film Reverse Transcription PCR (RT-PCR) • Used if RNA produced is below detection level for Northern blot • Isolate RNA from tissue of interest • Convert into double stranded cDNA • Amplify by PCR • Run on agarose gel
- 68. Studying Gene Expression Real Time PCR (qPCR) • Eliminates the need for running agarose gels • Is quantitative
- 69. Studying Gene Expression Gene microarray Microarray animation
Editor's Notes
- Congratulations! You finished a very challenging crash course in molecular biology! In chapter 3 we are going to see how everything you learned in chapter 2 is applied in the field of biotechnology.
- In module one you watched a video to learn how to pour and load an agarose gel. Agarose gel electrophoresis is the most basic and common technique used in a biotechnology lab. Agarose is a gel matrix that separates DNA and RNA based on size, shape and charge.
- Remember from module one that the backbone of DNA is made up of sugar and phosphate molecules that are held together by phosphodiester bonds. The phosphate molecules on the DNA backbone are negatively charged. These phosphate molecules confer a negative charge on the entire DNA molecule. Therefore, because opposites attract, when placed in an electrical field the negatively charged DNA will migrate toward the positive pole.
- Because all DNA is negatively charged, regardless of the length or source, the rate of DNA migration and separation through an agarose gel depends on the size of the DNA molecule. Agarose provides a gel matrix through which the DNA molecules migrate. Smaller molecules travel faster through the gel matrix than larger molecules. Think about it this way, if you pulled a series of nets across the center of a pool and released a tank full of snakes and worms in one end of the pool, the smaller worms will easily fit through the holes in your net and will get to the other end of the pool faster than the larger snakes which will get tangled in the net. It’s the same for DNA molecules. The smaller molecules are better able to navigate through the matrix and therefore travel faster than larger DNA molecules.
- Click on the electrophoresis animation to visualize agarose gel electrophoresis.
- Now that you understand the most common technique used in a biotechnology lab, let’s learn about the most rapidly developing field in biotechnology, recombinant DNA technology. Recombinant DNA technology, or genetic engineering, allows DNA to be combined from different sources. Do you remember when we mentioned in module one that the GFP gene from jellyfish has been inserted in expressed in animals such as this fish. When the GFP gene is expressed, the fish glow green under UV light. You may have also seen in the news recently that scientists in South Korea have created the first transgenic dog. This Beagle puppy expresses a protein found in sea anemones that causes the dog to glow red under UV light. The applications for recombinant DNA technology are limitless.
- Before we can understand the applications of recombinant DNA technology, we must first understand the definition of recombinant. DNA, whether or linear (as pictured on the left) or circular (such as plasmid DNA pictured on the right), can be used as a vector for transferring genes to organisms. Recombinant DNA is simply DNA that has taken up a foreign piece of DNA as is shown in the bottom diagrams. Well, how do you get this foreign DNA into the vector? Good question. We first have to understand the function of restriction enzymes.
- A restriction enzyme is a protein that was originally discovered in bacteria. Because bacteria do not have an immune system, bacteria use restriction enzymes as a defense mechanism against invading viral DNA. When the phage virus shown in the diagram releases it’s DNA into the bacterial cell, the restriction enzymes bind to the viral DNA and cut it into little pieces. This defense protects bacterial cells from destruction by invading viruses, but it can be exploited by biotechnologists to create recombinant DNA. Let’s learn how.
- Restriction enzyme binds to and cut at a specific DNA sequence, called the recognition site. Notice that the name of this restriction enzyme is EcoRI and it’s recognition site is GAATTC. The restriction enzyme works by breaking the phosphodiester bonds that join adjacent nucleotides in a DNA strand.
- After cutting a sample of DNA with restriction enzymes, you can separate the fragments by agarose gel electrophoresis. Remember what we learned about the fragments migrating through the matrix in an electric field? That’s right! The smaller fragments migrate the fastest and larger molecules migrate slower and will be closer to the well at the end of the electrophoretic run.
- Click on the animation to visualize a restriction digest separated by agarose gel electrophoresis.
- Most restriction enzymes bind to and cut within the recognition site. Some restriction enzymes cut straight down the middle of the recognition site, but some restriction enzymes cut in a zig zag pattern. Notice that this zig zag pattern generates overhangs that are complementary to one another. These are called sticky ends.
- Restriction enzymes that produce sticky ends are useful for recombinant DNA technology because DNA from different sources can be cut with the same restriction enzyme and the overhanging sticky ends will complementarily base pair. This is how you create a recombinant DNA molecule. DNA ligase seals the nicks in the phosphodiester backbone between the vector DNA and newly inserted fragment of DNA.
- Click on the animation to visualize the production of a recombinant DNA molecule created by a restriction enzyme digest.
- OK. So we’ve learned how to insert a gene into DNA to create recombinant DNA. Well then, how do you get that DNA into a whole organism, like the glowing Beagle puppy? To understand this, you need to understand transformation, the process by which organisms take up and express foreign DNA. In module one we learned that Frederick Griffith demonstrated the first transformation in an experiment with a pathogen of mice. Click on the animation to refresh your memory about the data recovered from Griffith’s experiment.
- Bacterial cells have the ability to take in and express DNA from their surrounding environment. The bacterial cell will replicate and express this inserted DNA just as it replicates and expresses genes on it’s chromosomal DNA.
- Scientists can create recombinant plasmids by using restriction enzymes to insert a gene of interest into a plasmid vector. That recombinant plasmid vector can then be introduced to bacterial cells that have been treated for transformation. One reason for transforming bacterial cells is to replicate the recombinant gene of interest. This is called gene cloning. The generation or doubling time for different strains of bacteria vary, but the average doubling time for bacteria used in transformation experiments is 20 minutes. That means that every 20 minutes the number of bacteria (and therefore the number of copies of the recombinant DNA) are doubled. Transformation of recombinant plasmid DNA into bacterial cells is a simple and inexpensive way to culture large amounts of the DNA you are interested in. Click on the gene cloning animation to visualize this application of bacterial transformation.
- Sometimes researchers screen a library for a specific gene. In shotgun cloning, the entire genome is cloned and sequenced and then individual genes are sorted out later through bioinformatics. Bioinformatics is an interdisciplinary field that applies computer science and information technology to promote an understanding of biological processes.
- In shotgun sequencing, overlapping sequence fragments are aligned using computer programs to assemble entire chromosomes.
- After a gene is cloned, it is important to determine the nucleotide sequence of the gene. That means to determine the order of the G’s, A’s, T’s and C’s in that segment of DNA. This is a picture of a sequencing gel. How do you create and read one of these?
- Well first we need to review DNA replication. It’s been a while and we need to refresh our memories. Remember that before a cell divides it must make a new copy of DNA for the new cell. To do that, the parental strands must be separated, a primer attaches to each parental strand and zooming in on the replication diagram we can see that DNA polymerase extends from the primer by adding nucleotides that are complementary to the parent strand. The type of bond that holds the nucleotides together in the growing strand is called a phosphodiester bond. Zooming in even further on the replication diagram we can see that the phosphodiester bonds, shown in yellow, are formed between the phosphate group on one nucleotide and the OH group on the adjacent nucleotide.
- Frederick Sanger developed a method to sequence DNA and therefore this method is referred to as the Sanger sequencing method. This method uses dideoxynucleotides in a sequencing reaction. Notice that dideoxynucleotides are almost the same as deoxynucleotides. There is one very important difference. There is no OH group on the third carbon. The removal of that OH makes it impossible to form phosphodiester bonds. That means polymerase will not be able to extend the growing strand of new DNA. The chain will end with the incorporation of the dideoxynucleotide.
- Let’s look inside a typical Sanger sequencing reaction. Notice that to set up these reactions, you need four separate reaction tubes. The components in all of the tubes are the necessary components for DNA replication (single stranded template DNA, primer, DNA polymerase, and normal deoxynucleotides for extending the growing chain). The difference between the four tubes is the addition of the dideoxynucleotide. The first tube has dideoxy-T, but it also has regular deoxy-T’s. That means that as polymerase adds T’s, sometimes it will add a regular T and the chain will continue to grow. But sometimes polymerase will add a dideoxy-T and cause the chain elongation to terminate. Because there are lots of copies of the template DNA, over time there will be a dideoxy-T incorporated at all positions in the newly synthesized strands, generating all possible subfragments ending in T of the template strand of DNA. In this short example, the fragments ending in T will be 7 base pairs or 2 base pairs. The same reaction is occurring in all four tubes, but because a different dideoxynucleotide was used in each tube, the subfragments ending in each corresponding nucleotide will be generated. The fragments ending in A 8 base pairs, 3 bp, or 1 bp. Look at the tube containing dideoxy-C. The fragments ending in C will be 9 bp, 5 bp, or 4 bp. And in the last tube, the fragments ending in G will be 6 bp. These fragments can be separated on a gel.
- Each tube is loaded into a different well on the gel. Remember what we learned earlier in the module about the speed of DNA fragments in an electrical field? That’s right! Larger fragments run slower than small fragments. That means when you run the samples on the gel, they will separate out by size. You can read the gel from the bottom to the top to tell you the sequence of the template strand of DNA you sequenced. That means the sequence of this DNA molecule is ATACCGTAC.
- Click on the animation to visualize a sequencing reaction.
- patents
- The Sanger method can only be used to sequence approximately 300 bases at a time. Because of this limitation, Sanger sequencing is rapidly being replaced by computer-automated sequencers. This method uses a single reaction tube and the dideoxynucleotides are each labeled with a different fluorescent dye. Samples are separated by electrophoresis and scanned with a laser beam. The laser stimulates the fluorescent dyes, creating color patterns for each nucleotide. The fluorescence pattern is processed and converted by a computer to generate the sequence of the template DNA. The Human Genome Project was developed as an international scientific research project with a primary goal to determine the sequence of nucleotide base pairs which make up all human DNA. The Human Genome Project was completed 5 years ahead of schedule largely due to the development of automated sequencing technology.
- Applications for medicine
- Because we share many of the same genes as these organisms, complete genome sequences of these model organisms have been incredibly useful for comparative genomics studies that allow researchers to study gene structure and function in these organisms in ways designed to understand gene structure and function in humans. Applications for developmental and evolutionary biologists.
- PCR was developed in the mid 1980’s by Kerry Mullis and it is really just DNA replication in a tube. So the requirements for a PCR reaction are the same as the requirements for DNA replication. You must add the template DNA, which can often be total genomic DNA. The target is the region on the template that you wish to amplify. You need to add primers, which are short single stranded molecules of DNA that flank the target region. Just like in replication, the primers allow DNA polymerase to attach and begin replication. A special DNA polymerase is added, called Taq DNA polymerase. This polymerase acts just like any other DNA polymerase in that it attaches nucleotides to the newly growing strand of DNA by matching the complementary nucleotide on the template strand. Taq DNA polymerase is special because it was discovered in a bacteria that grows in a hot spring. That means that it can withstand extremely high temperatures. We’ll explain why that is important in just a minute. If Taq DNA polymerase is going to extend the growing strand of DNA, there must be deoxynucleotides in the reaction mix. These are the G’s, A’s, T’s and C’s that polymerase incorporates into the new strand.
- Once all of the components have been added to the reaction tube, the reactions are placed in a machine called a thermal cycler. The thermal cycler is an instrument that is capable of rapidly changing temperature over very short time intervals. The machine repeatedly cycles through three different temperatures during a PCR reaction. Let’s learn about these temperature cycles.
- The first temperature is 94 degrees celsius. This is called the denaturation step because the hydrogen bonds between the double stranded DNA are broken, or denatured, making all of the DNA single stranded.
- The temperature is then dropped to around 55 degrees celsius. This is called the annealing step because it allows the single stranded primers to find and attach to their complement on the template DNA. Remember that the primers were created to specifically flank the target region you desired to amplify.
- The third step is to raise the temperature to 72 degrees celsius. This step is called the elongation step because at this temperature the Taq DNA polymerase is extending from the primer by adding nucleotides that are complementary to the template to the newly growing strand of DNA. At the end of this third temperature step, you have completely replicated the target region of DNA and that means you have doubled the number of copies of the target.
- The thermal cycler literally cycles through these three temperature steps for the number of times programmed into the machine, usually around 30 times.
- Every time the machine cycles through the temperatures, the number of copies of the target is doubled. That means after one cycle you will have two copies, then four copies, then 8, 16, 32….and so on. Because of this logarithmic amplification, PCR is an easy way to generate lots of copies of a desired region of DNA and has numerous applications in biotechnology.
- Click on the PCR animation to visualize this process.
- Gel electrophoresis is remarkably useful. One problem with gel electrophoresis is that it is not a permanent copy of the data. DNA continues to diffuse through the agarose matrix even after you stop the electrophoretic run. You can take a picture to keep a permanent record, but you cannot manipulate the DNA in any way. One way to create a permanent record of the restriction digest that can be tested and manipulated is to transfer and fix the DNA onto a nylon membrane. Edward Southern was the scientist who developed this technique, so it is now referred to as a “Southern Blot”. Human genomic DNA is 3 billion base pairs and contains thousands of recognition sites for restriction enzymes. When human genomic DNA is cut with restriction enzymes and run on an agarose gel you usually see a smear like the picture on the left. Transferring this DNA onto a nylon membrane allows researchers to hybridize specific probes to generate pictures like the one on the left. This picture is much clearer and more informative. Let’s learn how this southern blot hybridization is done. A Southern blot is used to determine if a specific DNA sequence is present in a sample or where a band containing a specific sequence is located on a gel.
- As mentioned in the last slide, the first step in preparing a southern blot is to cut genomic DNA with restriction enzymes. The restriction enzymes will bind to and cut within it’s recognition site all along the 3 billion base pairs of genomic DNA. These restriction digests are then loaded onto an agarose gel and separated in an electrical field.
- The DNA is then denatured in a basic solution. That means the hydrogen bonds holding the double stranded helix will be broken and the DNA will be single stranded. The single stranded DNA is then transferred onto a nylon membrane. The DNA is crosslinked onto the membrane with UV light or with heat. This creates a permanent copy of the DNA that was separated on the agarose gel.
- You may have wondered why the DNA had to be made single stranded before it was transferred to the membrane. Well, that was so you could hybridize a probe to the transferred DNA. A DNA probe is a short, single stranded molecule that is labeled (either radioactively or fluorescently). Because both the probe and the transferred DNA are single stranded, when incubated together they complementarily base pair. When the probe binds to it’s complement on the DNA it is called hybridizing the probe.
- Because the probe is radioactively labeled, it can be exposed onto a large sheet of x-ray film. After exposure to the film you will not see the long smears of genomic DNA, you will only be able to visualize the bands where the probe hybridized to the DNA on the membrane.
- Click on the Southern Blot Animation to visualize this process.