Laboratory. Extraction of DNA

Transcription

1 Laboratory 8 Extraction of DNA

2 Biology 171L FA17 Lab 8: Extraction of DNA Student Learning Outcomes - Learn the basic structure, components, and function of DNA. - Extract DNA from a fruit. - Practice using SNPs to determine % similarity among species. - Learn the basics of peer-review. Relevant Readings Campbell Biology, Chapter 16, especially pp A Short Guide to Writing about Biology, Chapter 6 (Revising) Homework Synopsis (see pages 8-11 & 8-12 for full description) Part I Mastering Biology Part II Science Communication Peer review of a classmate s first draft of photosynthesis paper Part III Data Analysis SNP short answer questions INTRODUCTION Proteins play a critical role in a cell, taking part in almost all living processes and reactions. Proteins are polymers, long chains of amino acids strung together by peptide bonds. Amino acids are the basic units that make up proteins and are structurally similar, sharing an amino group and carboxyl group but differing in their side chain, designated R. The size and polarity of the side chain, R, lends unique chemical properties to each amino acid. Although there are over 70 Fig. 1: Example of peptide chain amino acids naturally occurring, only 20 amino acids are utilized by living organisms to create proteins. These twenty amino acids can be used to create diverse peptide strands. The peptide chains fold in order to create the three dimensional globular structure of proteins. Each amino acid in the peptide chain may affect how the chain folds and ultimately affect part of the final protein shape. In Lab 6, you worked with a protein where shape was critical for protein function and cell health. The effect of the protein enzyme, catalase, on the decomposition of hydrogen peroxide was analyzed. Hydrogen peroxide is toxic to living cells. However, both animal and plant cells naturally produce hydrogen peroxide as an unfortunate by-product of metabolism. Cells have developed a self-preservation mechanism to render harmless this toxic by-product. In animal cells, the hydrogen peroxide producing reactions are segregated into membrane-bound organelles to contain the H 2 O 2. Plant cells produce the enzyme peroxidase and animal cells produce catalase. These enzymatic proteins have active sites that bind to H 2 O 2 and promote its Biol 171L - FA17 Extraction of DNA 8-2

3 breakdown. Changes to the amino acid within the enzyme s peptide chains could alter the shape of the active site. Changes to the active site could interfere with its ability to bind to hydrogen peroxide and inhibit the enzyme. Instead of breaking down, the hydrogen peroxide could increase until high levels cause cell damage or death In Lab 5, you studied the catalytic respiration of sugars in yeast. The sugar, lactose, was not utilized as a food source because yeast lacks the protein enzyme, betagalactosidase. Beta-galactosidase breaks down lactose and allows it to be used as a food source. The Fig. 2: Specificity of active site shape bacterium Escherichia coli (E. coli) is able to produce beta-galactosidase enzymes in response to levels of lactose in its environment. Thus E. Coli bacteria are able to thrive in the presence of lactose. Once the lactose has been consumed, E. Coli stops producing beta-galactosidase proteins, conserving energy for other living functions. Every living cell must be able to synthesize an enormous number of proteins. DNA, deoxyribonucleic acid, is the chemical record of those instructions. A DNA molecule is a double-stranded double helix composed of linear sequences of nucleotide units. Within the DNA strand are stretches of nucleotides that code for the sequence of Fig. 3: DNA double helix amino acids that comprise a particular peptide strand. That coded stretch is called a gene. Each living cell contains the genes that determine an organism s traits and this information is transmitted from one generation to the next. Eukaryotes have a nucleus, surrounded by a phospholipid bilayer membrane called the nuclear membrane. DNA is found in the nucleus. DNA is also found in the mitochondria within each cell. In humans, mitochondrial DNA is only passed on from mothers to all offspring. Your mitochondrial DNA is an identical match to your mother s mitochondrial DNA (barring random mutations). Prokaryotes do not have a true nucleus and thus their DNA is located in the cell cytoplasm. The size of a DNA strand is 100,000 times larger than the size of a cell, but only takes up 10% of the volume. If the DNA molecules from just one single human cell were stretched from end to end, the length would be over 1 meter. All that material is contained within the nucleus, which is typically just 2-4 micrometers in diameter. DNA molecules also have a slightly negative charge, which makes it difficult to compress into a small space. In order to fit the DNA into a prokaryotic cell or eukaryotic nucleus, DNA is tightly wrapped around histones, positively charged proteins. Together, the DNA and proteins found in the chromosomes of eukaryotes are called chromatin. The chromatin compacts even further to form chromosomes. Every cell in an organism has an identical set of chromosomes; thus every cell* carries ALL the genetic Biol 171L - FA17 Extraction of DNA 8-3

4 information it will ever need. *Red blood cells do not have DNA. The number of chromosomes differs between organisms but does not always reflect complexity. Humans have 23 pairs of chromosomes for a total of 46 chromosomes. Dogs have 39 pairs for a total of 78 chromosomes. Fig. 4: Karyotype of human DNA Human DNA consists of 23 unique chromosomes; 22 are called autosomes and the 23rd chromosome is a sex chromosome, either an X or Y chromosomes. Humans are diploid organisms, meaning that they have two sets of each chromosome, one inherited from your mother and one from your father. Figure 5 is a karyotype, a profile showing the number and appearance of chromosomes, in a human nucleus. There are 22 pairs of autosomal chromosomes and a pair of sex chromosomes. This karyotype shows an X and Y chromosome for sex chromosomes. The presence of the Y chromosome identifies the subject as a male. This week s lab will be using strawberries for genetic extraction. Strawberries are ideal candidates for extraction, because they contain a large amount of genetic material. Strawberries have just 7 chromosomes, but are octaploids, that is, they have 8 copies of each chromosome. Strawberries are also ideal because the plant tissue is soft and is easy to pulverize. In order to extract the DNA found in the nucleus, the cells must be broken down and the DNA must be separated from the cellular material. DNA SEQUENCES A DNA strand is a linear sequence of nucleotides covalently bonded together. Nucleotides all have a central deoxyribose sugar, a phosphate group, and one of four nitrogen bases. The four nitrogen bases are adenine, thymine, cytosine, and guanine. These four nucleotides are used to form the long chains of DNA. Because the sequence of these nucleotides codes for the production of peptide strands, DNA is directional. DNA strands (and RNA strands) are read in one direction to translate the code. The sugars that make up the deoxyribose sugar are used as directional reference. The five carbons in the deoxyribose sugar in nucleotides are given prime designations. The phosphate group is attached on the 5` end of the nucleotide (bonded to the 5` carbon in the deoxyribose structure). The other end of the nucleotide is the 3` end, with the 3` carbon atom in the deoxyribose structure free to bond the phosphate group of another nucleotide and lengthening the DNA strand. Biol 171L - FA17 Extraction of DNA 8-4

5 Fig 5. Left side shows basic structure of a nucleotide unit with the 5` and 3` carbon atoms highlighted and the right side shows the four DNA nucleotide bases Every time a cell replicates (mitosis) the cell must make exact duplicates of all the chromosomes to pass on full sets to each of its two daughter cells. Sometimes, mistakes (mutations) happen during replication (copying of DNA). Some mutations may occur in vital areas that lead to cell fatality and those mutations cause cell death. But many mutations either cause non-lethal changes or have no effect at all. Those mutations will be passed on to all cells resulting from that cell dividing (cell lines). However, mutations that occur during meiosis, the creation of gametophores for the purpose of reproduction, have a larger influence because those mutations are passed on to the following generations. Over time, DNA sequences change or evolve as variations of genes are passed on and new variations are created through mistakes in replication. Measuring the number of mutations is an important technique that scientists use to test for relatedness among organisms and to test for gene disorders. A single substitution mutation at one base pair site on a gene is called a single nucleotide polymorphism (SNP). For the most part, species that have a low number of SNP s are more closely related than sequences that have a larger difference. Once DNA has been extracted, how do you get this type of data? Through multiple steps that you will learn about in other classes, the extracted DNA is prepared for sequencing. The first step in sequencing is to increase the amount of DNA extracted from your organism (it is hard to extract DNA from most organisms, especially if they are small, unlike the strawberries). Polymerase Chain Reaction (PCR) is a method used to replicate a specific sequence of DNA (Fig. 6). Briefly, organismal DNA is placed in a tube with many loose nucleic acids, DNA Polymerase, primers and other materials that help drive the reaction. Nucleic acids provide the building blocks to replicate the strands of DNA. DNA Polymerase is an enzyme that builds a complementary strand of DNA onto a single strand of DNA. Primers bind to the DNA at specific sites and activate DNA Polymerase that then duplicates DNA from your samples. When your mix is assembled, you put it in a thermocycler. This machine can change its temperature rapidly according to a program that you determine. Every time DNA is heated above 90 C it unravels and Fig. 6 DNA Replication Biol 171L - FA17 Extraction of DNA 8-5

6 becomes single stranded. Then, as the thermocycler cools (to approx. 70 C) the DNA Polymerase is activated, and attaches to the primer site, which builds a complementary strand to your template DNA. Every region of DNA that you have selected (by designing your primer) is duplicated in every cycle of high temperature. Using multiple cycles, we can exponentially duplicate the amount of DNA in the tube. Typical PCR methods have cycles and create thousands to millions of copies of the DNA you started with. The next step is to confirm that the DNA is of high quality, i.e., pure - free of proteins and other contaminants. Confirmation of DNA quality can be done multiple ways, but the most common and cheapest method is to conduct gel electrophoresis. Briefly, DNA is loaded onto an agarose gel and the gel is orientated so that there is a negative charge closest to the DNA and a positive charge on the opposite side of the gel. When the electricity is turned on, the DNA fragments travel through the gel towards the positive charge; this is because DNA are negatively charged molecules and they are attracted to the positive charge. However, the gel provides resistance, so the length of the DNA strand affects how far the DNA can travel through the gel. Using gel electrophoresis, you can separate DNA fragments of different sizes [# of base pairs (bp)], which is illustrated in the left hand column of Fig. 7. You can use a known size standard (called a ladder) to determine the length of your DNA (the band in Fig. 7 is approx. 700 bp long). Size Standards 3,500 bp 1,000 bp 100 bp - charge + charge Fig 7. Gel Electrophoresis DNA band If the DNA is concentrated enough and is the expected size, you have DNA that is appropriate for sequencing. When the presence of good quality DNA is confirmed, another PCR method is used to clean the sequences. The strands of DNA are prepared by attaching nucleic acids to the DNA that have fluorescent tags. In this way the sequencer can read every color on a strand and call the base pairs; this is called Sanger sequencing. Some laboratories have their own sequencer, however, most labs send their sequences to a facility that charges a fee (currently the facility at UH charges $1.50 a sequence). The sequencing facility sends a text file with every base pair in the sequence identified (what you will use in today s exercise). Once the DNA has been sequenced there are many different ways to use it. In today s lab we will use the sequences to determine how related two organisms are. We assume that organisms with fewer SNP s are more closely related. So once you know how similar/different your sequences are for multiple individuals, you can determine if they are the same species, or belong to the same genus, family, etc. If the sequence of your organism is in the Barcode of Life Data Systems (BOLD) you can use a basic local alignment search tool (Blast) to compare your sequence to a database that identifies the organism that it came from. There are multiple databases on line and the most commonly used one is GenBank, which is housed at NCBI: The advantage of the NCBI website is you can search Biol 171L - FA17 Extraction of DNA 8-6

7 many different gene regions. However, today we are going to use the BOLD database at; This database uses two commonly sequenced gene regions, cytochrome c oxidase subunit 1 (COI), and RuBisCO large subunit (RBCL). The most common gene region used for animals is COI. It is a gene that is found in mitochondria and has a relatively fast mutation rate (this is necessary to distinguish among species). The RBCL gene is found in chloroplasts where it codes for RuBisCO, an enzyme necessary for carbon fixation (photosynthesis) and is used to identify species of plants. If researchers discover an unknown organism they can use the BOLD website to identify their organism (this only works if the sequence for that organism is already in the database). This is the activity you will do today, you have been provided multiple sequences and you will count the number of SNP s and use the BOLD website to determine what species you have and how they are related. Preparation for Lab Read through Introduction Research topics and terms that you are not familiar with or do not fully understand Peptide and Protein Structure DNA structure Genes Polymerase Chain Reaction Single Nucleotide Polymorphisms Read through Experiment Procedure EXPERIMENT PROCEDURE: Part 1: Strawberry DNA Extraction 1. Obtain a piece of strawberry from your TA. 2. Close Ziploc bag and mash the strawberry firmly for 2 minutes, making sure that you DO NOT break the bag. 3. Using a plastic transfer pipe (white tape label) and the 15 ml falcon tube (small), carefully measure 10 ml of the 0.9% NaCl solution and add to bag. 4. Using a transfer pipet (pink tape label) and the 15 ml falcon tube (small), measure ~2 ml of 10% SDS solution into the bag. 5. Seal bag and continue to mash strawberry thoroughly for another 2 minutes. Filtering Debris 6. Assemble the filtration apparatus by setting up an empty 50 ml Falcon tube (big), a funnel, and a square of 4-ply cheesecloth. Biol 171L - FA17 Extraction of DNA 8-7

8 Fig. 7: Filtration apparatus set-up 7. Carefully strain the slush (filtrate) into the apparatus and let it drip into the falcon tube. 8. When it is finished straining the slush, remove the funnel from the falcon tube. 9. Discard the cheesecloth and strawberry debris into the trash receptacle designated by TA. 10. Ethanol for the next step is in the freezer ask your TA or TI for help. The colder the alcohol, the better the results. DNA is not soluble in cold ethanol. Obtain approximately 2mL for your extraction (or get 10mL for your table to share). 11. Slowly add the cold ethanol to the falcon tube containing strawberry filtrate. Do so by tilting the falcon tube and slowly pouring the ethanol down the side of the falcon tube. The goal is to form a layer of ethanol on top of the layer of strawberry extract. 12. The alcohol layer should form above the extract and the DNA should be visible as a clear mucus-like substance at the interface of the alcohol layer. DNA was in the extract solution (pink bottom layer). It has a low density so it was floating in the top portion of the extract layer. As it interacts with the cold ethanol at the interface, it is not soluble in ethanol and precipitates out of solution. 13. Let the mixture stand for 1 minute 14. Use a glass stirring rod to gently dip into the top of the strawberry extract solution and bring more DNA up into the ethanol layer. The DNA is attracted to plastic and glass and should cling to the rod. 15. Attempt to spool the DNA on the stirring rod and remove from tube. 16. Record observations. 17. Clean-up If you haven t already, discard cheesecloth/ Ziploc in class waste. Empty the contents of your tube into the liquid waste container located by the sink. Rinse the tube and funnel with tap water and leave to dry. Wipe down lab bench areas with ethanol Part 2: Analyzing Sequences A. Count SNPs 1. Each table has been provided with 2 copies of sequence data. 2. Work in groups of two to count the number of SNP s in a sequence using the sequence on top as your reference. Biol 171L - FA17 Extraction of DNA 8-8

9 3. Divide the number of SNP s by the total number of base pairs in your sequence. This is your % difference. 4. Do this for each sequence on your data sheet. B. Compare Sequences 5. Compare your answers with the other group at your table. Did you get the same % difference as the other group? If not make sure you check your data; it is critical to have an accurate % difference. 6. Record this data in your notebook; you will use it in future labs. C. Identify your sequences 7. Once your group has calculated the % difference of each sequence, you will identify the species you are studying. 8. Download the text file from Laulima that corresponds with your group # as indicated at the top of your data sheet. 9. Copy the text for each sequence individually. Use a text editor application (e.g., WordPad). Do not use MS Word to open the file; MS Word attaches formatting to a text file that interferes with the website. 10. Go to the BOLD website: CHOOSE THE CORRECT TAB for the gene region for your sequences (COI is for animals, and RBCL is for plants) 12. Paste your sequence data into the box on webpage. Click Submit. 13. On the output screen you can scroll down and see a table with the top 20 hits. Choose the species data on top of the table; this is the closest match to the sequence you submitted. Fill in the following table for each of your sequences: Your Group: Sequence # Family Genus Species Similarity % Part 3: Peer Review Peer review has three primary goals: 1. To provide a way to help you make improvements to your paper, so that you can turn in your best possible work to your instructor for grading. 2. To provide an opportunity for you to learn from your peers; and Biol 171L - FA17 Extraction of DNA 8-9

10 3. To help you gain practice in critically reviewing written work, so that you can apply those skills to your own writing. Peer review, like most skills, improves with practice. We will be doing an in-class exercise where you will get to practice reviewing and commenting on a piece of student work. Your TA will give you further instructions in class. Biol 171L - FA17 Extraction of DNA 8-10

11 Lab 8 Homework Due Week of October 23, 2017 Format is important. All written homework should be typed, double-spaced, Times New Roman 12-pt font, with 1-inch margins. Remember to include your name, section and the name of your TA. Part 1 Mastering Biology (31 points): Answer the questions in the assignment entitled 9. Mendelian Genetics on the Mastering Biology site. You have until the night before lab at 11:59PM to complete these questions. Part 2 Science Communication (20 points): A. Peer Review (20 pts) Peer review is an important part of how scientists improve their science and their writing. This week you will review one of your classmate s papers. Using the peer review rubric (posted on Laulima) and Pechenik, 2013 (A Short Guide to Writing about Biology, Chapter 6, especially pp ), it will be your task to help your classmate improve their paper, and one of your classmates will do the same for your paper. Answer the questions that are provided; the questions will help you to develop your critical reading skills. Read the paper with a view to helping your classmate improve organization, flow and logic. You can be especially helpful by pointing out problems with figures and/or tables (e.g., captions in the wrong location). Do not spend too much time on spelling and grammar your classmates (and you!) will have an opportunity to fix these problems later on. See the rubric posted on Laulima for point distribution. Type your comments using 12-pt font, Times New Roman font, double-spaced with 1 margins. You must turn in two (2) copies of each part of this exercise: i.e., the lab report that you put comments on; and, the questions you answered in the peer review rubric!! Part 3 Data Analysis (20 points) Answer the following questions about your group s species: 1. From the BOLD results, were you able to definitively identify each of your five sequences? Why or why not? What evidence is listed? (5 pts) Biol 171L - FA17 Extraction of DNA 8-11

12 2. How are your organisms related? Are they all the same species, in the same genus, in the same family? Describe their taxonomic relationships. (5 pts) 3. On the BOLD website, there is table of the closest hits. Use the #1 sequence from your group and determine the top five sequences that match your sequence. Write their family, genus, and species names, and their % similarity score. (5 pts) Group: Sequence: Species: Order Family Genus Species Similarity % 4. Pick one species that your group identified in class and describe its ecology. What does it eat, where does it live (include the type of habitat and geographic range), is it endangered, does it have novel adaptations that protect it from predators? (5 pts) Biol 171L - FA17 Extraction of DNA 8-12