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From Genetics to DNA

Extracting DNA modified from:

Edible model of DNA:







Students will:

• Build a model of DNA

• Discuss the mechanisms for translation

• Examine simulated sequences of DNA

• Extract, cleave the proteins, isolate and precipitate out DNA, making it visible

• Simulate restricting DNA

• Simulate running this DNA in gel electrophoresis

• Determine which of the three suspects in this simulation is implicated

• Examine a variety of pictures of gels to determine paternity, and eliminate or implicate suspects in a crime scene

Benchmarks:

Life Science

CCG Organisms: Understand the characteristics, structure, and functions of organisms.

SC.03.LS.01 Recognize characteristics that are similar and different between organisms.

SC.05.LS.01 Group or classify organisms based on a variety of characteristics.

SC.05.LS.01.01 Classify a variety of living things into groups using various characteristics.

CCG Heredity: Understand the transmission of traits in living things.

SC.03.LS.03 Describe how related plants and animals have similar characteristics.

SC.05.LS.04 Describe the life cycle of an organism.

SC.05.LS.04.01 Describe the life cycle of common organisms.

SC.05.LS.04.02 Recognize that organisms are produced by living organisms of similar kind, and do not appear spontaneously from inanimate materials.

SC.08.LS.03 Describe how the traits of an organism are passed from generation to generation.

SC.08.LS.03.01 Distinguish between asexual and sexual reproduction.

SC.08.LS.03.02 Identify traits inherited through genes and those resulting from interactions with the environment.

SC.08.LS.03.03 Use simple laws of probability to predict patterns of heredity with the use of Punnett squares.

CCG Diversity/Interdependence: Understand the relationships among living things and between living things and their environments.

SC.08.LS.05 Describe and explain the theory of natural selection as a mechanism for evolution.

SC.08.LS.05.01 Identify and explain how random variations in species can be preserved through natural selection.

Materials:

• Permanent

▪ Scissors

▪ Copies of DNA

▪ Copies of DNA sequencing

▪ Copies DNA codes for proteins

▪ Blender

▪ Ice bucket

▪ Measuring cup

▪ 1/8 Measuring spoon

▪ Strainer

▪ Clean empty milk jug (for water)

▪ Copies of gel electrophoresis results

▪ Gel for electrophoresis model

▪ Copies of gel photographs

▪ DNA models (optional)

• Consumable

▪ 4 red licorice sticks

▪ 2 black licorice stick

▪ color mini marshmallows

▪ white mini marshmallows

▪ wax paper

▪ toothpicks

▪ Color paper for copies of proteins

▪ Copies of DNA sequence

▪ Copies of restriction enzymes

▪ Cups

▪ Centrifuge tubes

▪ Isopropyl alcohol (at least 95%)

▪ Ice

▪ Salt

▪ Liquid dish soap

▪ Unseasoned meat tenderizer

▪ Dried peas

▪ Sterile swabs

▪ Glue sticks

▪ Masking tape or small labels

▪ Permanent markers – fine point

▪ Copies of Amino Acid circles

Discussion:

This lesson plan is part two of three-parts in understanding DNA and how DNA fingerprints work. In the genetics lab, your students looked at variation in individuals to cement their foundation for understanding the how (alleles) behind individual variation. In this class, there are 4 activities to help students conceptualize DNA both as a model and actually seeing it, and that variation is caused by a change in the sequence. In addition, students will model how scientists use DNA sequences in solving crimes. Examining gels displaying mother, child and potential fathers reinforce student understanding of genetics from the prior lesson.

Overview of Activities:

Activity 1

Students will continue their understanding of DNA through building an edible model of the DNA, an edible mRNA, and build a protein from the mRNA. The structure of DNA is discussed, being comprised of 4 bases, Adenine, Cytosine, Guanine, and Thymine, and the AT and GC pairing of them. The sequences of these four bases are a code that translates into amino acids, building proteins that enable a cell to function properly. There are sequences of DNA that are highly conserved (i.e. 16S and 16S rRNA), and portions of these sequences are identical from bacteria to us. These code for very important functions, like protein building.

Activity 2

Students will extract and isolate DNA from their cheek cells by breaking membranes, cleaving histones, allowing the cellular debris to settle, and precipitating the DNA in a layer between water and ice cold isopropyl alcohol, concentrating it so that it is visible to the naked eye. This extraction-cleaving-isolation-precipitation is done using common household materials.

Activity 3

There are sections in our DNA that do not code for any gene. These are referred to as “junk” DNA. There is a lot of variation in the DNA in these regions. In fact there is so much variation, we can use them to identify individuals. Students then examine paper sequences for 3 individuals and a crime scene. The students add a paper restriction enzyme that cleaves the DNA in specific locations. The students then run their paper DNA out on a table gel electrophoresis, and determine which of the three is the culprit.

Activity 4

Students practice what they have learned by examining photographs of different crime scene gels. From these gels, they can identify the culprit. In addition, students can examine paternity gels to identify that the alleles come from both their mother and their father.

Set-up for Class:

Students work with a partner, but everyone participates with their own analysis

• Set out for each pair of students:

▪ copies in page protectors of DNA

▪ copies in page protectors of sequencing DNA

▪ copies in page protectors of DNA codes for proteins

▪ copies of Crime Scene DNA gels

▪ copies of DNA paternity gels

▪ ~36 toothpicks

▪ 2 large sheets wax paper

▪ 2 pair scissors

▪ color disk “amino acids”

▪ Student work copies of Crime Scene DNA sequences

▪ Student work copies of Crime Scene restriction enzymes

▪ 2 Student “gel”

▪ 2 cups (3 ounce)

▪ 2 centrifuge tubes

▪ permanent markers

▪ 2 sterile swabs

• Put in convenient location but away from students:

▪ Red licorice

▪ Black licorice

▪ White mini marshmallows

▪ Color mini marshmallows

▪ Masking tape (or little labels)

▪ DNA extraction chemical center

o Salt

o Dish washing soap

o Meat tenderizer

o Bucket with ice

o Isopropyl alcohol buried in the ice

DNA Structure Activity 1

Supplies:

• Per student:

▪ 2 pieces of red licorice

▪ 17 toothpicks

▪ 9 pink marshmallows (Thymine)

▪ 9 white marshmallows (Uracil)

▪ 18 yellow marshmallows (Cytocine)

▪ 18 green marshmallows (Adenine)

▪ 18 orange marshmallows (Guanine)

▪ Masking Tape or small labels

▪ Scotch Tape

▪ Permanent markers – fine point

• 1 piece black licorice

• 2 of each of the Amino Acid circles

Discussion:

Structure

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA molecules is the long-term storage of information and DNA is often compared to a set of blueprints, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.

Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription. Most of these RNA molecules are used to synthesize proteins, but others are used directly in structures such as ribosomes and spliceosomes.

Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms such as animals, plants, and fungi store their DNA inside the cell nucleus, while in prokaryotes such as bacteria it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA, which helps control its interactions with other proteins and thereby control which genes are transcribed.

Physical and chemical properties

DNA is a long polymer made from repeating units called nucleotides. The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Ångstroms (0.33 nanometres) long. Although each individual repeating unit is very small, DNA polymers can be enormous molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, is 220 million base pairs long.

In living organisms, DNA does not usually exist as a single molecule, but instead as a tightly-associated pair of molecules. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base, which interacts with the other DNA strand in the helix. In general, a base linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. If multiple nucleotides are linked together, as in DNA, this polymer is referred to as a polynucleotide.

The backbone of the DNA strand is made from alternating phosphate and sugar residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a strand of DNA has a direction. In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand. This arrangement of DNA strands is called antiparallel. The asymmetric ends of DNA strands are referred to as the 5′ (five prime) and 3′ (three prime) ends. One of the major differences between DNA and RNA is the sugar, with 2-deoxyribose being replaced by the alternative pentose sugar ribose in RNA.

The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are shown below and are attached to the sugar/phosphate to form the complete nucleotide, as shown for adenosine monophosphate.

These bases are classified into two types; adenine and guanine are fused five- and six-membered heterocyclic compounds called purines, while cytosine and thymine are six-membered rings called pyrimidines. A fifth pyrimidine base, called uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine, but a very rare exception to this rule is a bacterial virus called PBS1 that contains uracil in its DNA. In contrast, following synthesis of certain RNA molecules, a significant number of the uracils are converted to thymines by the enzymatic addition of the missing methyl group. This occurs mostly on structural and enzymatic RNAs like transfer RNAs and ribosomal RNA.

The double helix is a right-handed spiral. As the DNA strands wind around each other, they leave gaps between each set of phosphate backbones, revealing the sides of the bases inside (see animation). There are two of these grooves twisting around the surface of the double helix: one groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove.

Base pairing

Each type of base on one strand forms a bond with just one type of base on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. In a double helix, the two strands are also held together via forces generated by the hydrophobic effect and pi stacking, which are not influenced by the sequence of the DNA. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds (see figures, left). The GC base pair is therefore stronger than the AT base pair. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA. Long DNA helices with a high GC content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands. Parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in bacterial promoters, tend to have sequences with a high AT content, making the strands easier to pull apart. In the laboratory, the strength of this interaction can be measured by finding the temperature required to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt, the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.

Sense and antisense

A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is complementary to the sense sequence and is therefore called the "antisense" sequence. Since RNA polymerases work by making a complementary copy of their templates, it is this antisense strand that is the template for producing the sense messenger RNA. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction made above between sense and antisense strands by having overlapping genes. In these cases, some DNA sequences do double duty, encoding one protein when read 5′ to 3′ along one strand, and a second protein when read in the opposite direction (still 5′ to 3′) along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome. Another way of reducing genome size is seen in some viruses that contain linear or circular single-stranded DNA as their genetic material.

Supercoiling

The secondary structure of DNA is the helices. DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases. These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.

Mutations

DNA can be damaged by many different sorts of mutagens. These include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and x-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light mostly damages DNA by producing thymine dimers, which are cross-links between adjacent pyrimidine bases in a DNA strand. On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, as well as double-strand breaks. It has been estimated that in each human cell, about 500 bases suffer oxidative damage per day. Of these oxidative lesions, the most dangerous are double-strand breaks, as these lesions are difficult to repair and can produce point mutations, insertions and deletions from the DNA sequence, as well as chromosomal translocations.

Many mutagens intercalate into the space between two adjacent base pairs. Intercalators are mostly aromatic and planar molecules, and include ethidium, daunomycin, doxorubicin and thalidomide. In order for an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. These structural changes inhibit both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators are often carcinogens, with benzopyrene diol epoxide, acridines, aflatoxin and ethidium bromide being well-known examples. Nevertheless, due to their properties of inhibiting DNA transcription and replication, they are also used in chemotherapy to inhibit rapidly-growing cancer cells.

Overview of biological functions

DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation which depends on the same interaction between RNA nucleotides. Alternatively, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles; here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

Genome structure

Genomic DNA is located in the cell nucleus of eukaryotes, as well as small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the expression of the open reading frame.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences. The reasons for the presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma." However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.

Transcription and translation

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (43 combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA, TGA and TAG codons.

Tertiary Structure of DNA

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins found in chromatin include the high-mobility group proteins, which bind preferentially to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into more complex chromatin structures.

In contrast, other proteins have evolved to specifically bind particular DNA sequences. The most intensively studied of these are the various classes of transcription factors, which are proteins that regulate transcription. Each one of these proteins bind to one particular set of DNA sequences and thereby activates or inhibits the transcription of genes with these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter; this will change the accessibility of the DNA template to the polymerase.

As these DNA targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that mediate responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases, allowing them to "read" the DNA sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible.

Directions:

• Students work with a partner. Each partner needs to select a different sequence. The sequences are included in the picture of the double helix picture in the page protector.

▪ One partner needs to pick:

o DNA Sequence 1 – T A C G T A T G A A A C

▪ The other partner needs to pick:

o DNA Sequence 2 – T G G T T T A G A A T T

• To assemble one side of the DNA molecule:

▪ A piece of red licorice will form the backbone and marshmallows will be the chemical bases.

▪ Place a marshmallow on the end of a toothpick so that the point of the toothpick goes all the way through.

▪ Anchor the toothpick into the licorice backbone. Refer to the table with the sequence information for the color of marshmallow that represents the chemical bases in your sequence.

▪ Label the backbone. With a marker or pen and small stickers, label your licorice backbone “DNA- 1” or “DNA-2” depending on which sequence you used. Write the label on the left end of the licorice.

▪ Match the chemical base pairs. Place the color marshmallow for the matching chemical base on the other end of each toothpick. Remember that A always pairs with T and C always pairs with G!

▪ To complete your DNA model, attach the other backbone (red licorice stick) so your model looks like a ladder.

▪ Carefully twist your DNA molecule so that it looks like a double helix.

Transcription:

• The four chemical bases in DNA (A, C, G, and T) create a code. Cells “read” this DNA code to make proteins, the building blocks of all organisms. This is done in two steps:

▪ Copying the directions – Transcription

▪ Reading the copy to string together the small molecules (amino acids) that make up a protein – Translation.

• Making a Copy of DNA – Transcription

▪ Cells read DNA in small portions (genes) to create a protein. To do this, the cell must first make a copy of the gene’s code to send to the protein-building machinery. This process is called transcription. Using the following materials, follow the steps below to see how this is done.

• Transcription

▪ RNA is single stranded and only “reads” DNA from one strand.

▪ Unzip your DNA. In order to make this copy, the chemical bases forming the rungs of the DNA ladder must be separated. Do this by cutting or breaking in the middle the toothpicks in your model to separate the chemical bases and unzip the DNA ladder. Break the toothpicks between the two marshmallows.

▪ Set aside the unlabeled backbone (with chemical bases attached) aside.

▪ Begin to form your mRNA strand. The exposed chemical bases of the unzipped DNA are used to make the copy. This copy is called messenger RNA (mRNA). The mRNA molecule is also made of a backbone and the same chemical bases as DNA. There is one exception however – instead of Thymine (T), mRNA uses Uracil (U). The chemical bases in mRNA form pairs in the same way as DNA:

o Adenine (A) binds with Uracil (U)

o Guanine (G) binds with Cytosine (C).

▪ Place your backbone labeled “DNA-1” or “DNA-2” (depending on which you used to make your model) in front of you. Follow the rules of base pairing to make your mRNA copy of the DNA code by lining up colored marshmallows with their appropriate match. The chemical bases of mRNA are also attached to a backbone as in DNA.

▪ Attach the new chemical bases to a piece of black licorice backbone using toothpicks cut or broken in half. This forms a new mRNA copy of your DNA strand.

▪ • Label this new strand mRNA-1 or mRNA-2 (the same number as your DNA strand) on the left end of the backbone.

• Translation

▪ The mRNA copy of DNA is essentially a recipe for assembling a protein. Proteins are built from small molecules called amino acids. When the mRNA copy is sent to the protein-building machinery it is read and the appropriate amino acids are assembled. This process is called translation.

▪ Begin to create your protein. mRNA is read in groups of three chemical bases. Each group of three tells the cell which amino acid to assemble. In other words, each group of three is a “code” for a particular amino acid.

▪ You and your partner will need to work together to build your complete protein. Between the two of you, you will have both the mRNA-1 and mRNA-2.

▪ Place both strands of mRNA end-to-end on the table in front of you, with the mRNA-1 strand on the left.

▪ Look at the first 3 chemical bases on the left end of your mRNA strand.

▪ Use the Amino Acid Key to determine which amino acid these 3 chemical bases code.

▪ Place the colored circle cut-out representing that amino acid on the table directly below the three chemical bases.

▪ Repeat Step 1 for each group (or code) of three chemical bases on the mRNA strand.

▪ Now you have a protein!

▪ You may take home your DNA and mRNA models. Yummy!

Extracting DNA Activity 2

Supplies:

• Craft sticks or swab (1 per student)

• Cup (students can use the cup for their marshmallows from Activity 1)

1) Salt

• Water

• Liquid dish soap

• Ice cold 95-99% isopropyl alcohol

• Centrifuge tubes

• Permanent marker

Discussion:

Extracting DNA, amplifying DNA, using restriction enzymes and cleaving DNA are different techniques which scientists use to study evolution, medicine, and forensic science (to name just three). The focus of this lesson is the location of DNA within the cell. Cells are enclosed in membranes. In order to release the DNA, these membranes need to be broken without damaging the DNA. We do this with soap. Think about why you use soap to wash dishes or your hands. To remove grease and dirt, right? Soap molecules and grease molecules are made of two parts:

• Heads, which like water.

• Tails, which hate water.

Both soap and grease molecules organize themselves in bubbles (spheres) with their heads outside to face the water and their tails inside to hide from the water. When soap comes close to grease, their similar structures cause them to combine, forming a greasy soapy ball. A cell's membranes have two layers of lipid (fat) molecules with proteins going through them. When soap comes close to the cell, it captures the lipids and proteins. After adding the detergent, what do you have in your pea soup? (Free floating DNA from the nucleus still with the attached histone proteins and mitochondria.)

Salt was added to the pea (or spit). DNA is negatively charged. The salt provides the DNA with positively charged sodium ions to help neutralize it.

We add the meat tenderizers to cut the proteins away from the DNA. The DNA is wrapped around histones, which are proteins organizing DNA.

There seem to be a wide variety of answers to exactly the mechanisms with precipitate the DNA, and I have never been much satisfied with them. Perhaps they just seem too short – like this is a trivial question. Here is the best answer that I have found:

• The alcohol is less dense than water, and DNA is also less dense than water. The DNA is neutrally buoyant in the alcohol. DNA is polar and alcohol is non-polar. The combination of these factors precipitate the DNA out of the alcohol, and keep it suspended at that layer.

• I cannot locate any reasoning behind the ice-cold alcohol. When I am asked, I tell the students that I haven’t found the answer yet. Questions drive science, and we discuss the process of science.

Directions:

1) Label your centrifuge tube with your initials.

2) Fill the centrifuge tube about 1/2 full of water.

3) Pour this into your 3 oz cup.

4) Using the swab, scrap the inside of your mouth, collecting loose cheek cells. Scrap hard enough to get as many as possible, but not hard enough to hurt yourself.

5) Using the water in your 3 oz. cup, swish the water in your mouth, and spit it back into your cup.

6) Form a little spout with the cup and pour the water and spit/cheek cell solution into the centrifuge tube.

7) Put the swab into the tube and swish to remove the cells from the swab into the water.

8) Add a pinch of salt to the tube and gently swirl to mix. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see. (You can put on the lid of the tube, and GENTLY rock it back and forth.)

9) Add 2 drops of liquid soap and gently swirl to mix. Try not to form bubbles. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see. (You can put on the lid of the tube, and GENTLY rock it back and forth.)

10) Add a pinch of enzymes to the tube and gently swirl to mix. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see. (You can put on the lid of the tube, and GENTLY rock it back and forth.)

a) Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens cleaning solution.

b) In this experiment, meat tenderizer acts as an enzyme to cut proteins just like a pair of scissors.

11) Let the mixture sit for 10 minutes or more.

12) Tilt your test tube and slowly pour the ice cold alcohol (95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as your spit solution.

13) DNA will rise into the alcohol layer from the spit layer. You can use a wooden stick or other hook to draw the DNA into the alcohol.

a) Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide: "Hmmm...which layer should I go to?"

i) This is sort of like looking around the room for the most comfortable seat. Some will choose the couch, others might choose the rocking chair.

b) In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer.

c) DNA is a long, stringy molecule that likes to clump together.

• Now that you've successfully extracted DNA from one source, you're ready to experiment further. Try these ideas or some of your own:

▪ Experiment with other DNA sources. Which source gives you the most DNA? How can you compare them?

▪ Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?

▪ Experiment with leaving out or changing steps. We've told you that you need each step, but is this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient you use.

• Do only living organisms contain DNA? Try extracting DNA from things that you think might not have DNA.

Want to conduct more DNA extraction experiments? Try out different soaps and detergents. Do powdered soaps work as well as liquid detergents?

Gel Electrophoresis Modeling Activity 3

Supplies:

• Permanent

▪ Scissors

▪ Paper gel for electrophoresis model

▪ Copies of DNA sequence

▪ Copies of restriction enzymes – different color

▪ Glue sticks

Discussion:

DNA-modifying enzymes

Nucleases and ligases

Nucleases are enzymes that cut DNA strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of DNA strands are called exonucleases, while endonucleases cut within strands. The most frequently-used nucleases in molecular biology are the restriction endonucleases, which cut DNA at specific sequences. For instance, the EcoRV enzyme recognizes the 6-base sequence 5′-GAT|ATC-3′ and makes a cut at the vertical line. In nature, these enzymes protect bacteria against phage infection by digesting the phage DNA when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and DNA fingerprinting.

Because of their ability to cut DNA in predictable locations, they are commonly used in forensice science, as well as many other commonly techniques. [pic]

Electropherogram printout from automated sequencer showing part of a DNA sequence

A DNA sequence or genetic sequence is a succession of letters representing the primary structure of a real or hypothetical DNA molecule or strand, with the capacity to carry information.

The possible letters are A, C, G, and T, representing the four nucleotide subunits of a DNA strand - adenine, cytosine, guanine, thymine bases covalently linked to phospho-backbone. In the typical case, the sequences are printed abutting one another without gaps, as in the sequence AAAGTCTGAC, going from 5' to 3' from left to right. A succession of any number of nucleotides greater than four is liable to be called a sequence. With regard to its biological function, which may depend on context, a sequence may be sense or anti-sense, and either coding or noncoding. DNA sequences can also contain "junk DNA."

Genetic (or DNA) Fingerprinting

Forensic scientists can use DNA in blood, semen, skin, saliva or hair at a crime scene to identify a perpetrator. This process is called genetic fingerprinting, or more accurately, DNA profiling. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a criminal. However, identification can be complicated if the scene is contaminated with DNA from several people. DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case. People convicted of certain types of crimes may be required to provide a sample of DNA for a database. This has helped investigators solve old cases where only a DNA sample was obtained from the scene. DNA profiling can also be used to identify victims of mass casualty incidents.

Directions:

• Students work in groups of 4.

• Give each student one strip of DNA (from Suspect 1, Suspect 2, Suspect 3, or Crime Scene.

• Instruct each students to cut the DNA sequences and tape the for strips in sequence together to make one long strand.

• Students glue the restriction enzyme over the sequence whenever it matches. (Normally, the restriction enzyme will seek out the complimentary strand. However in this model, have the students match the sequence to eliminate any confusion. After this activity is completed, then we discuss that the restriction enzyme would actually bond to the complimentary DNA.)

• Students cut their strands where indicated on each of the restriction enzymes.

• Be very clear that the students MUST not contaminate their strands. They must keep them separate and identified.

• Direct the students to count the number of nucleotides in each fragment and writing this number on the back.

• The students fold their DNA to fit the well of the paper gel.

• Note that the paper gel already has a ladder.

• The students move their DNA fragments to the correct position (as indicated by the ladder).

• When completed, the gel should reveal which of the three suspects is the culprit.

Practice Reading Gels Activity 4

Supplies:

• Copies of DNA gel electrophoresis

Discussion:

Each gel is a mini mystery. By this time, students easily solve them. The paternity may take the younger students a bit longer, but refer them back to their Marshmallow bugs and Punnett squares. They receive only half of their genes from one parent. Half should match that parent. They receive the other half from their other parent. That have should match the other parent. Half of the parents, however, do not match the child. The child did not inherit those genes.

Directions:

• This is a quick – 2 to 15 minute activity.

• There are 5 pictures of crime scene gels,

• There is one nice diagram explanation paternity

• There are three paternity gels.

Note: There should be 6 copies of each of the gel photographs for the students. If you need to replace any of them, the documents: “paternity.doc” and “suspect.doc” Gail should have them.

| | |Second Position of Codon | | |

| | |T |C |A |G | | |

|F |T |TTT |TCT |TAT |TGT |T |T |

|i | |Phe |Ser |Tyr |Cys | |h |

|r | |[F] |[S] |[Y] |[C] |C |i |

|s | | | | | | |r |

|t | |TTC |TCC |TAC |TGC |A |d |

| | |Phe |Ser |Tyr |Cys | | |

|P | |[F] |[S] |[Y] |[C] |G |P |

|o | | | | | | |o |

|s | |TTA |TCA |TAA |TGA | |s |

|i | |Leu |Ser |Ter |Ter | |i |

|t | |[L] |[S] |[end] |[end] | |t |

|i | | | | | | |i |

|o | |TTG |TCG |TAG |TGG | |o |

|n | |Leu |Ser |Ter |Trp | |n |

| | |[L] |[S] |[end] |[W] | | |

| | | | | | | | |

| |C |CTT |CCT |CAT |CGT |T | |

| | |Leu |Pro |His |Arg | | |

| | |[L] |[P] |[H] |[R] |C | |

| | | | | | | | |

| | |CTC |CCC |CAC |CGC |A | |

| | |Leu |Pro |His |Arg | | |

| | |[L] |[P] |[H] |[R] |G | |

| | | | | | | | |

| | |CTA |CCA |CAA |CGA | | |

| | |Leu |Pro |Gln |Arg | | |

| | |[L] |[P] |[Q] |[R] | | |

| | | | | | | | |

| | |CTG |CCG |CAG |CGG | | |

| | |Leu |Pro |Gln |Arg | | |

| | |[L] |[P] |[Q] |[R] | | |

| | | | | | | | |

| |A |ATT |ACT |AAT |AGT |T | |

| | |Ile |Thr |Asn |Ser | | |

| | |[I] |[T] |[N] |[S] |C | |

| | | | | | | | |

| | |ATC |ACC |AAC |AGC |A | |

| | |Ile |Thr |Asn |Ser | | |

| | |[I] |[T] |[N] |[S] |G | |

| | | | | | | | |

| | |ATA |ACA |AAA |AGA | | |

| | |Ile |Thr |Lys |Arg | | |

| | |[I] |[T] |[K] |[R] | | |

| | | | | | | | |

| | |ATG |ACG |AAG |AGG | | |

| | |Met |Thr |Lys |Arg | | |

| | |[M] |[T] |[K] |[R] | | |

| | | | | | | | |

| |G |GTT |GCT |GAT |GGT |T | |

| | |Val |Ala |Asp |Gly | | |

| | |[V] |[A] |[D] |[G] |C | |

| | | | | | | | |

| | |GTC |GCC |GAC |GGC |A | |

| | |Val |Ala |Asp |Gly | | |

| | |[V] |[A] |[D] |[G] |G | |

| | | | | | | | |

| | |GTA |GCA |GAA |GGA | | |

| | |Val |Ala |Glu |Gly | | |

| | |[V] |[A] |[E] |[G] | | |

| | | | | | | | |

| | |GTG |GCG |GAG |GGG | | |

| | |Val |Ala |Glu |Gly | | |

| | |[V] |[A] |[E] |[G] | | |

| | | | | | | | |

Materials:

• Permanent

▪ Scissors

▪ Copies of DNA

▪ Copies of DNA sequencing

▪ Copies DNA codes for proteins

▪ Blender

▪ Ice bucket

▪ Measuring cup

▪ 1/8 Measuring spoon

▪ Strainer

▪ Clean empty milk jug (for water)

▪ Copies of gel electrophoresis results

▪ Gel for electrophoresis model

▪ Copies of gel photographs

▪ DNA models (optional)

• Consumable

▪ 4 red licorice sticks

▪ 2 black licorice stick

▪ color mini marshmallows

▪ white mini marshmallows

▪ wax paper

▪ toothpicks

▪ Color paper for copies of proteins

▪ Copies of DNA sequence

▪ Copies of restriction enzymes

▪ Cups

▪ Centrifuge tubes

▪ Isopropyl alcohol (at least 95%)

▪ Ice

▪ Salt

▪ Liquid dish soap

▪ Unseasoned meat tenderizer

▪ Dried peas

▪ Sterile swabs

▪ Glue sticks

▪ Masking tape or small labels

▪ Permanent markers – fine point

▪ Copies of Amino Acid circles

Gel Electrophoresis Chamber

50

45

40

35

30

25

20

15

10

5

You and your partner select different sequences below.

• DNA Sequence 1: T A C G T A T G A A A C

-or-

• DNA Sequence 2: T G G T T T A G A A T T

DNA:

o Adenine (A) binds with Thymine (T)

o Guanine (G) binds with Cytosine (C).

RNA:

o Adenine (A) binds with Uracil (U)

o Guanine (G) binds with Cytosine (C).

Marshmallow Table

|Nucleic Base |Color of Marshmallow |

|Adenine |Green |

|Thymine |Pink |

|Guanine |Orange |

|Cytosine |Yellow |

|Uracil |White |

AMINO ACID KEY

|Code |AAA |ACC |ACU |AUG |CAU |UAA |UCU |UUG |

|Amino Acid |Red |Orange |Orange |Yellow |Green |Blue |Purple |White |

| | | | |(start) | |(stop) | | |

Suspect 1

|T |A |T |G |C |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

EcoRV

|G |A |T |A |T |C |

Materials:

• Permanent

▪ Scissors (in teacher kit)

▪ Copies of DNA

▪ Copies of paper Electrophoresis Carriage

▪ Copies of DNA sequencing

▪ Copies DNA codes for proteins

▪ Copies of crime scene gels

▪ Clean container (for water)

▪ Copies of color amino acids

▪ Small Ice Chest

• Consumable

▪ 24 red licorice sticks (or same color twizzle sticks)

▪ 12 black licorice stick (or different color twizzle sticks)

▪ color mini marshmallows

▪ white mini marshmallows

▪ wax paper

▪ toothpicks

▪ Copies of DNA sequence

▪ Copies of restriction enzymes

▪ 3 oz Cups

▪ Centrifuge tubes

▪ Isopropyl alcohol (at least 95%)

▪ Ice

▪ Salt

▪ Liquid dish soap

▪ Unseasoned meat tenderizer

▪ Sterile swabs or craft sticks

▪ Glue sticks

▪ Masking tape or small labels

▪ Permanent markers – fine point )

-----------------------

Minor Groove

Major Groove

Marshmallow Toothpick Licorice Stick

White Yellow White

White Yellow White

White Yellow White

White Yellow White

White Yellow White

White Yellow White

White White Orange

White White Orange

White White Orange

White White Orange

White White Orange

White White Orange

White Green Green

White Green Green

White Green Green

White Green Green

White Green Green

White Green Green

Yellow Green White

Yellow Green White

Yellow Green White

Yellow Green White

Yellow Green White

Yellow Green White

Green White Orange

Green White Orange

Green White Orange

Green White Orange

Green White Orange

Green White Orange

Green Yellow White

Green Yellow White

Green Yellow White

Green Yellow White

Green Yellow White

Green Yellow White

Green Yellow Yellow

Green Yellow Yellow

Green Yellow Yellow

Green Yellow Yellow

Green Yellow Yellow

Green Yellow Yellow

Green Green Green

Green Green Green

Green Green Green

Green Green Green

Green Green Green

Green Green Green

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