New Yorks Community Biotech Lab

Biotechnology Crash Course

January 2012 Ellen D. Jorgensen, Ph.D., Instructor

Biotechnology is actually a collection of many different technologies, all of which use cells and biological molecules to solve problems and make useful products. One of the turning points of modern science was the advent of genetic engineering. Recombinant DNA technology facilitated this. The term recombinant DNA refers to the joining or recombining genetic material from two different sources. This happens sometimes in nature, and is desirable because it results in an increase genetic variation. However, recombinant DNA can also be made artificially, in the laboratory. This was first made possible by the discovery of restriction enzymes. Restriction enzymes allowed scientists to cut DNA into small, well-defined pieces that were easy to work with. The pieces can be visualized using a technique known as gel electrophoresis, in which DNA molecules are separated by size. Another key technology was the ability to amplify DNA via PCR (short for polymerase chain reaction). This powerful molecular biology technique specifically amplifies a single or a few copies of a piece of DNA by several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It was developed in the 1980s and is now one of the most widely-used technologies in biotech. It is used both in constructing recombinant DNA and also for exploration of genetics and genomics. In this crash course we will cover DNA structure & function, and get hands-on training in the major techniques of biotechnology including the use of enzymes to cut and splice DNA, PCR, gel electrophoresis, and inserting foreign DNA into bacteria. The two projects we will complete are 1) using mitochondrial DNA from our cheek cells to do a genotyping experiment related to ancestry and 2) genetic engineering of the bacterium E. coli.

Course Schedule
Session 1: Lecture: DNA structure, genetic dogma, restriction enzymes Plasmid Construction: Restriction Enzyme Digestion of Lamba or pBR322 DNA Gel Electrophoresis

Session 2: Lecture: PCR Tracking Human Origins via Mitochondrial DNA: DNA Extraction from Cheek Cells PCR Analytical Electrophoresis, send samples out for sequencing

Bacterial Transformation with premade plasmids

Session 3: Tracking Human Origins Via Mitochondrial DNA: Analysis of Sequence data

Plasmid Construction: Plasmid preps and analytical electrophoresis

Basic Techniques

Measuring Devices
In biotechnology we routinely use metric volumes and weights. 1 microliter (1uL) is 1/1000,000 Liter 1 milliliter (1 mL) is 1/1,000 Liter Therefore 1000uL = 1 mL Concentrations of solutions are expressed either by their molarity (a measurement of concentration based on the number of molecules present) or as a percentage (weight/volume, weight/weight, or volume/volume). Biomolecules such as enzymes are often provided in mg/mL (milligram per milliliter) solutions. For large volumes (greater than 25mL) we will use graduated cylinders to measure liquids. For volumes in the 1-25 mL range there are calibrated disposable plastic pipets that fit into a handheld electronic dispenser. However, we will also be working with very small volumes in microcentrifuge (microfuge) tubes. For these we will use a micropipettor, often called a pipetman. These are mechanical devices that use displacement to deliver volumes between 1 and 1,000 microliters. We will be using three models that differ in the range of volumes they can accurately deliver.

P-1000 delivers 200-1000uL P-200 delivers 20-200 uL P-20 delivers 1-20uL The photo shows typical pipetmen of the three major sizes. Note the difference in the barrel sizes. The displays only accommodate 3 digits (equivalent to a 1% error in the largest measurement). So the P-1000 display will read 100 when set to deliver 1000uL and the P-20 will read 100 when set to deliver 10.0 uL. So you have to be very aware when using these devices and double-check that your volumes are properly set before using. And dont forget to put on a clean tip!

Using Micropipets
Most micropipets (often called a pipetman) have a two-position plunger with friction stops. The plunger is at the top of the pipetman. Your thumb operates the plunger with the remaining fingers of your hand wrapped around the barrel. Depressing to the first stop measures the desired volume. Depressing to the second stop introduces an additional volume of air to blow out any solution remaining in the tip. A smaller button on top engages a metal frame around the pipetman that ejects the used tip when pushed. Always be sure that a fresh disposable plastic tip is on the end of the pipetman before using, and that is it appropriate for the range of the pipetman. P-1000s use the largest tips (usually blue in color) while P-200s and P-20s use a smaller tip that is usually yellow. P-10s and P-2s have their own tips which are the smallest, but we rarely use these in our labs. Apply a little pressure when using the barrel to pick the tip up. The tip should fit snugly on the white barrel of the pipetman. When withdrawing or expelling fluid, always hold your sample tube firmly between thumb and forefinger. Hold tube at nearly eye level so you can observe fluid level change in pipet tip. Do not try to pipet with your tube in a rack or try to pipet into a tube held by lab partner. To Withdraw Sample: Depress plunger to first stop and hold in this position. THEN dip tip into solution to be pipetted and SLOWLY draw fluid into tip by releasing plunger upwards. Slide pipet tip out along inside wall of tube to dislodge any excess droplets adhering to outside of tip. To Expel Sample: Touch pipet tip to inside wall of tube into which you want to empty sample. This creates a capillary effect that helps draw fluid out of tip. If the amount to be pipetted is very small, and you are pipetting into a tube that already holds some liquid, you may dip the tip of the pipetman into the solution before delivering the sample. Slowly depress plunger to first stop. Depress to second stop to blow out last bit of fluid. Hold plunger in depressed position. Slide pipet out of tube with plunger depressed to avoid sucking liquid back into tip. To prevent cross-contaminating reactions, always use a fresh tip for each reagent and each reaction tube.

Restriction Enzymes
Restriction enzymes are proteins produced by bacteria to prevent or restrict invasion by foreign DNA. They act as DNA scissors, cutting the foreign DNA into pieces so that it cannot function. Restriction enzymes recognize and cut at specific places along the DNA molecule known as restriction sites. Each different restriction enzyme (and there are hundreds, made by many different bacteria) has its own type of site. In general, a restriction site is a 4- or 6- basepair sequence that is a palindrome. A DNA palindrome is a sequence in which the top strand read from 5 to 3 is the same as the bottom strand read from 5 to 3. For example, 5 GAATTC 3 3 CTTAAG 5 is a DNA palindrome and is the restriction site for the enzyme EcoRI. The name EcoRI comes from the bacterium in which it was first discoveredEscherichia coli RY 13 (EcoR) and I, because it was the first restriction enzyme found in this organism. EcoRI makes one cut between the G and the A in each of the DNA strands (see below). After the cuts are made, the DNA is held together only by the hydrogen bonds between the four bases in the middle. Hydrogen bonds are weak, and the DNA comes apart. Cut DNA: 5 G AATTC 3 3 CTTAA G 5 As you can see, when EcoRI cuts a DNA molecule it leaves single-stranded tails on the new ends. This type of end is called a sticky end because it is easy to rejoin it to complementary sticky ends. Not all restriction enzymes make sticky ends; some cut straight across the DNA molecule, producing a blunt end. When scientists study a DNA molecule, one of the first things they do is figure out what restriction sites are present, and where the restriction sites are in the DNA molecule. A restriction enzyme digest of a particular DNA molecule produces a distinctive pattern of DNA fragments, specific to that DNA molecule, which can then be seen with gel electrophoresis. Molecular biologists use restriction enzymes to prepare fragments of DNA containing regions of interest (like genes) for subsequent splicing together to form new constructs. Heres a good video of the process: http://www.youtube.com/watch?v=piGOuud3f40

Gel Electrophoresis
Electrophoresis means literally to carry with electricity. It is a widely-used biotechnology technique used to separate charged molecules such as DNA, RNA, and proteins. Electrophoresis is frequently performed using an agarose gel. Agarose is a polysaccharide similar to pectin that dissolves in boiling water and then gels as it cools. In electrophoresis, the sample is applied to a slab of gelled agarose and then an electric current is applied across the gel. Agarose gels must be prepared and run in a buffer. The buffer is necessary because ions would otherwise cause the anode to become alkaline and the cathode to become acidic. The buffer we will use is tris-borate-EDTA (TBE). A buffer is a mixture of a weak acid or base and its salt. Tris is a weak base, and the Tris salt is made by adding boric acid. The metal chelator EDTA (ethylenediaminetetraacetic acid) is also added. By chelating (specifically binding to) calcium ions, EDTA inhibits RNases and DNases, which could degrade RNA and DNA. It is important to have the proper concentration of buffer. In the absence of ions (e.g., if the gel were mistakenly run in water) electrical conductance is minimal. On the other hand, if the buffer is too concentrated (e.g., if 10 buffer were used by mistake) electrical conductance is too efficient and heat is generated. Too much heat will melt the gel and denature the DNA. When an electric field is present, a negatively charged sample (such as DNA) migrates through the gel toward the positive electrode. The rate of migration of a DNA molecule depends on the size and also on whether the molecule is circular or linear. The voltage applied to the agarose gel also influences how quickly a sample moves through the gel. The higher the voltage, the more quickly the sample moves. However, there is a tradeoff because at higher voltages samples do not separate with as much resolution (i.e. the bands are not sharp and well-defined). Also, if the voltage is too high, the gel will become hot and melt. This is a photo of an agarose gel showing bacterial plasmid DNA that was cut with restriction enzymes and electrophoresed. DNA was loaded into the slots visible at the top of the photo. The current was applied so that the DNA migrated from top to bottom, so the bands nearest the bottom represent smaller pieces of DNA. For an explanation and an interactive demo of electrophoresis, go to the website: http://learn.genetics.utah.edu/content/labs/gel/

Bacterial Transformation
Transformation in this case means to trick the organism into accepting foreign DNA into its genetic material. In bacteria, the process happens naturally by several different means and is a way to diversify the organism. Still, it is rare for most bacteria to take up DNA naturally from the environment. But by subjecting bacteria to certain artificial conditions, we can enable many of them to take up DNA. When cells are in a state in which they are able to take up DNA, they are referred to as competent. Making cells competent usually involves changing the ionic strength of the medium and heating the cells in the presence of positive ions (usually calcium). This treatment renders the cell membrane permeable to DNA. More recently, high voltage has also been used to render cells permeable to DNA in a process called electroporation. Once DNA is taken into a cell, the use of that DNA by the cell to make RNA and proteins is referred to as expression. In nature, the expression of the newly acquired DNA depends upon its being integrated into the DNA of the host cell. As discussed above, the process of integration is known as recombination, and it requires that the new DNA be very similar in sequence to the host genome. However, researchers usually want to introduce into a cell DNA that is quite different from the existing genome. Such DNA would not be recombined into the genome and would be lost. To avoid this problem, scientists transform host cells with plasmid DNA. A plasmid is a small, circular piece of double-stranded DNA that has an origin of replication (see http://homepages.strath.ac.uk/~dfs99109/BB211/Plasmidnotes.html for a good explanation of plasmids). An origin of replication is a sequence of bases at which DNA replication begins. Because they contain origins of replication, plasmids are copied by the host cells DNA replication enzymes, and each daughter cell receives copies of the plasmid upon cell division. Therefore, plasmids do not need to be recombined into the genome to be maintained and expressed. Additionally, since plasmids do not have to have DNA that is similar to the host cells DNA, DNA from other organisms can be maintained as a plasmid. Fortunately, it is relatively easy to introduce new DNA sequences into plasmids. Plasmids naturally occur in bacteria and yeast, and they are widely used as vehicles for introducing foreign DNA into these organisms. Thus far, no analogs of plasmids are known for higher plants and animals, which is one reason why genetic engineering is so much more difficult in higher organisms. We will use plasmid DNA to transform E. coli K12 and give it new attributes such as colored fluorescence or chemiluminescence. In order to transform bacteria using plasmid DNA, biotechnologists must overcome two problems. Typically, cells that contain plasmid DNA have a disadvantage since cellular resources are diverted from normal cellular processes to replicate plasmid DNA and synthesize plasmid-encoded proteins. If a mixed population of cells with plasmids and cells without plasmids is grown together, then the cells without the plasmids grow faster.

Therefore, there is always tremendous pressure on cells to get rid of their plasmids. To overcome this pressure, there has to be an advantage to the cells that have the plasmid. Additionally, we have to be able to determine which bacteria received the plasmid. That is, we need a marker that lets us know that the bacterial colony we obtain at the end of our experiment was the result of a successful gene transfer. To accomplish both goalsmaking it advantageous for cells to retain plasmids, and having a selectable marker so we can recognize when bacteria cells contain new DNAwe will use a system involving antibiotics and genes for resistance to antibiotics. This system is a powerful tool in biotechnology. In a typical transformation, billions of bacteria are treated and exposed to plasmid DNA. Only a fraction (usually fewer than 1 in 1000) will acquire the plasmid. ntibiotic resistance genes provide a means of finding the bacteria which acquired the plasmid DNA in the midst of all of those bacteria which did not. If the plasmid sed to transform the DNA contains a gene for resistance to an antibiotic, then after transformation, bacteria that acquired the plasmid (transformants) can be distinguished from those that did not by plating the bacteria on a medium containing the antibiotic. Only the bacteria that acquired the plasmid will overcome the killing effect of the antibiotic and grow to form colonies on the plate. So the only colonies on an antibiotic plate after a transformation are the bacteria that acquired the plasmid. This procedure accomplishes our two goals of giving an advantage to cells that have a plasmid so the plasmid is retained and of having a marker so we know our cells contain new DNA. Resistance to an antibiotic is known as a selectable marker; that is, we can select for cells that contain it. Of course, in this case we will see the chemiluminescence or the fluorescent color of the transformed bacteria as well. But without the selectable marker this population would be in the tiny minority and would be out-competed by the untransformed cells.

Polymerase Chain Reaction (PCR)

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based analysis of genes, the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. Mullis was awarded the Nobel Prize in Chemistry for his work on PCR. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and short pieces of DNA called primers, which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands of the DNA double helix at a high temperature in a process called DNA melting. At the lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. . As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. This YouTube video illustrates the process quite nicely (but turn off the sound if you hate technopop): http://www.youtube.com/watch?v=HMC7c2T8fVk

Appendix

Double Helix Structure of DNA

The four nucleotide bases of DNA

Base-pairing involves hydrogen bonds, which are much weaker than covalent bonds. In the center of the helix, the A (adenosine) residues are attracted to T (thymidine) and G (guanine) to C (cytosine). They fit together like magnets, and the fit is quite specific. You can see that the A-T base pair has only two hydrogen bonds, while the G-C has three. That gives it a different geometry so mispairing is unfavorable. Those three bonds also make G-C stronger than A-T, a property that makes long GC-rich runs of DNA difficult to sequence.

When DNA is copied in a cell in the natural process of cell division, the major protein responsible is the enzyme DNA polymerase. It uses deoxyribonucleotide triphosphates (dNTPs) as building blocks to make a new copy of the DNA strands. We take advantage of this process in PCR, where the DNA polymerase from a heat-loving bacterium is used to amplify DNA artificially. When DNA from a gene is transcribed into an RNA copy called messenger RNA (mRNA), the enzyme responsible is RNA polymerase. There are many other cofactors necessary, but the polymerase is the protein responsible for creating the nascent RNA chain. It uses ribonucleotide triphosphates (rNTPs) as building blocks to make an mRNA chain. Translating mRNA into protein is the most difficult step. A giant complex of proteins and structural RNAs called a ribosome is responsible for this process. It uses adaptor molecules called transfer RNAs (tRNAs) to shuttle amino acids into the proper order to produce the polypeptide chain of a newly synthesized protein.

The amino acids coded for by DNA triplets (codons)

Restriction Map of pBR322

Really good videos of biological processes:

DNA replication and packaging, Transcription and translation: http://www.youtube.com/watch?v=4PKjF7OumYo&feature=related Invasion of a bacterial cell by bacteriophages (viruses that only infect bacteria): http://www.youtube.com/watch?v=ehbZpo8oXSs&feature=related Restriction enzyme EcoR1 cutting DNA: http://www.youtube.com/watch?v=aA5fyWJh5S0