Answered: - Finished the first part of this lab (except the last question).

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Answered: - Finished the first part of this lab (except the last question).

Finished the first part of this lab (except the last question). Need the rest finished.

BIOL1408 Introductory Biology

Dr. Flo Oxley

Name

date

Lab 12. DNA & RNA Lab Report

This lab unit will not use the custom eSciences ACC Lab Manual. You may need to purchase some supplies from

your local grocery or drug store to supplement the materials found in your kit to complete this exercise, in which you

will isolate DNA from food(s).

BACKGROUND INFORMATION: DNA STRUCTURE

The genes that encode the information for making all of the proteins in your cells are located in DNA, a nucleic acid

found in your chromosomes. DNA molecules are very long thin fibers made of a double-stranded helix. The nucleotides are held together within each strand by a covalent bond called a ?phosphodiester? bond. The DNA structure is a

double helix of two strands of DNA. The two strands are held together by much weaker forces called ?hydrogen

bonds?.

As you can see from these images, the DNA double helix has two DNA strands made up of alternating phosphate and

deoxyribose groups. Nitrogenous bases (Adenosine or A, Guanine or G, Thymine or T, and Cytosine or C) are are

hydrogen-bonded to each other in the interior of the double helix. The hydrogen bonds are between specific nitrogenous bases on the opposite strands: G hydrogen bonds to C, and A hydrogen bonds to T. The specificity of these

base-pairings allow for the accurate replication of the DNA sequence by DNA polymerase during cell division.

Notice that there are 2 hydrogen bonds between each A & T base pair, and 3 hydrogen bonds between each G & C

base pair. While it is hard to break apart the nucleotides along each of the DNA strands, it doesn?t take too much to

separate the two strands from each other. High temperatures (around the boiling point of water) is enough to separate strands, or the ?denature? them. At cold temperatures, however, the DNA double helix is a stable structure.

DNA will denature (the two strands come apart) at high temperatures, but the greater number of hydrogen bonding

between G & C base-pairs means that GC-rich DNA will denature more slowly at high temperatures.

Notice that the distance spanning the base-pairs in the inside of the double helix is constant: the base-pairs are between one pyrimidine (C & T have one ring in their nitrogenous base) and one purine (A & G have two rings in their

nitrogenous bases).

Notice also that the two strands of DNA run in opposite directions, or as said to be ?antiparallel? to each other. You

can see the opposite directions by looking at the labeling of the strands in the figures above: one strand runs from 3?

to 5?, while the opposite strand runs from 5? to 3?. Alternatively, you can see the directionality by examining the deoxyribose residues of the phosphate-sugar backbone of each strand. Notice that there are 4 carbons and one oxygen in each of the deoxyribose residues. The oxygen is on the top of one strand of DNA, and at the bottom of the

other strand of DNA in the double helix. This antiparallel orientation will play an important role in the replication of

DNA, as we will see in the PCR DNA Fingerprinting at the end of this lab exercise.

DNA Structure Background Questions

To better understand the structure of DNA and how it works in the cell, you might want to read the chapter on DNA in

your textbook (Chapter 7) and go to: http://learn.genetics.utah.edu/content/basics/ to watch some animations from

their ?Tour of the Basics? library.

1. Which DNA bases pair with each other?

2. Look at the strands of DNA in the photo above. The pentagonal structure with an oxygen (O) in the ring is a ribose. Each DNA strand has a phosphoribosyl backbone with a nitrogenous base facing towards the interior of the

DNA double helix. Notice that the direction of the two strands is opposite to each other.

a. Describe how you can tell from the appearance of the pentagonal ribosyl structures that the

direction of the two strands are opposite to each other.

b. Follow one of the strands from top to bottom. How is the top end numbered, and how is the

bottom end numbered? How does this compare with the numbering of the complementary

strand?

3. How is genetic information passed on through generations without change?

4. How many different types of nucleotides are found in DNA?

5. How many different types of amino acids are found in proteins?

6. How do genes encode information for amino acid sequences of proteins? Be specific!

7. If you could pull all of the DNA from one of your cells and stretch them DNA molecules end-to-end, how far would

your cell?s DNA stretch?

8. How many chromosomes are in each of your cells? Why do they come in pairs? Why are there 2 sex chromosomes?

Experiment 1: DNA Extraction

In this lab, you isolate DNA from the interface an ice-cold fruit extract and ice-cold alcohol, by spooling the DNA as it

precipitates there. It also is not difficult to take a solution of DNA and separate the DNA molecules from the solvent.

DNA is poorly soluble in alcohols such as ethanol and isopropanol. DNA solubility is reduced even more when the

solution has a high salt concentration and the alcohol is very cold.

In a process referred to as ?spooling DNA?, ice-cold alcohol is carefully over-layered on top of an ice-cold DNA solution. At the interface of the alcohol and the DNA solution, DNA molecules encountering the alcohol come out of solution and form an insoluble membrane-like layer between the two phases. Because the DNA molecules are so long

and fibrous, they tend to entangle each other at this interface, such that if you very carefully snag the membrane with

a glass rod and twist it, you can actually wind up or ?spool? the entangled DNA molecules around the rod. If you twist

the rod slowly and gently enough, you can end up spooling enough DNA that you can actually see and feel it after

you bring the rod out.

spooling DNA

Experiment 1: DNA Extraction Protocol

This is a simple, effective protocol for spooling DNA. Ripe strawberries are an excellent source for extracting DNA be?

cause they are easy to pulverize and contain enzymes called pectinases and cellulases that help to break down cell

walls. And most important, strawberries have eight copies of each chromosome (they are octoploid), so there is a lot

of DNA to isolate.

NOTE: If you prefer a slightly more elaborate protocol for spooling DNA, this is available at: http://learn.ge?

netics.utah.edu/content/labs/extraction/howto/ . The protocol found at this site isolates DNA from split peas,

and requires the use of a kitchen blender.

The purpose of each ingredient in the DNA extraction procedure is as follows:

Shampoo or dishwasher soap (helps to dissolve the cell membrane, which is a lipid bilayer.)

Sodium chloride (helps to remove proteins that are bound to the DNA. It also helps to keep the proteins dis?

solved in the aqueous layer so they don?t precipitate in the alcohol along with the DNA.)

Ethanol or isopropyl alcohol (causes the DNA to precipitate. When DNA comes out of solution it tends to

clump together, which makes it visible. The long strands of DNA will wrap around the stirrer or transfer pipet

when itis swirled at the interface between the two layers.)

Notes on Materials and Recipes

? Use Ziploc freezer bags rather than sandwich bags, as they are thicker.

? Fresh or frozen strawberries can be used. Be sure to thaw the frozen berries at room temperature. Bananas or kiwi

fruit can also be used but yield less DNA.

? Use non?iodized table salt or laboratory?grade sodium chloride.

? 95% ethanol or 91 or 100% isopropyl alcohol can be used to precipitate the DNA. Isopropyl alcohol can be pur?

chased from a pharmacy. Whichever you use, make sure it is ice cold by placing in an ice?water bath or in the

freezer.

NOTE: An alternative protocol calls for all of the ingredients described below, except frozen

strawberries are used and the pulverization is done in a blender.

DNA Extraction Buffer

? 1 ml (1/4 teaspoon) shampoo (without conditioner) or 0.5 ml (1/8 teaspoon) dishwasher detergent

? 0.15 grams sodium chloride (a pinch)

? water to 10 milliliters (mL)

DNA Isolation from Strawberries

Student Directions

Materials

? 1?3 strawberries (about the volume of a golf ball). Frozen strawberries should be thawed at room temperature.

? 10 ml DNA Extraction Buffer (soapy salty water, recipe above)

? about 20 ml ice cold 91% or 100% isopropyl alcohol (chilled in the freezer for at least 30 minutes)

? 1 Ziploc freezer bag

? 1 clear test tube, or shot glass

? 1 funnel lined with a moistened paper towel or coffee filter

? 1 coffee stirrer or transfer pipet

Directions

1. Remove the green sepals from the strawberries.

2. Place strawberries into a Ziploc freezer bag and seal tightly.

3. Squish for a few minutes to completely squash the fruit.

4. Add 10 ml DNA Extraction Buffer (soapy salty water, recipe abave) and squish for a few more minutes. Try not to

make a lot of soap bubbles.

5. Filter through a moistened paper towel set in a funnel, and collect the liquid in a clear tube. Do not squeeze the pa?

per towel. Collect liquid into a test tube or shot glass.

6. Carefully add 2 volumes ice cold isopropyl alcohol to the strawberry liquid in the tube. Pour the isopropyl alcohol

slowly down the side of the tube so that it forms a separate layer on top of the strawberry liquid without mixing into the

strawberry liquid. Because the alcohol has a lower density than water solutions, the alcohol layer will naturally stay

on top unless you bump or mix the layers.

7. Watch for about a minute. What do you see? You should see a white fluffy cloud at the interface between the two

liquids. That?s DNA!

8. Spin and stir the coffee stirrer or transfer pipet in the tangle of DNA at the water/alcohol interface, wrapping the

DNA around the stirrer.

9. Pull out the stirrer and transfer the DNA to a piece of saran wrap or clean tube. If you have any ?Blue Dye? left over

from Experiment 6. Diffusion and Osmosis, add a drop to the DNA on your saran wrap and mix with a tooth pick.

What do you see? The blue dye stains DNA, and the fibers that you see are actually thousands and millions of DNA

strands pulled together in the fiber.

10. Rinse your funnel. Put the Ziploc TM bag and paper towel in the garbage.

Experiment 1. DNA Extraction Questions

1. Why did we use a salt in the extraction solution? High salt makes DNA less soluble in water. In order to dissolve, the water needs to interact with the DNA. Since DNA is quite large, it needs to interact with lots of water for this purpose. When you add salt, the water preferentially interacts with the salt (it's small, and can

move around in solution easier than the DNA can). This makes it so there is less water available to interact

with the DNA and it becomes less soluble.

2. What else might be in the ethanol/aqueous interface? How could you eliminate this? By stirring the solutions together, mixing the two.

3. What is the texture and consistency of DNA? DNA is viscous and slimy. Very similar to snot / mucous

4. Is the DNA soluble in the aqueous solution or alcohol? Yes

5. If you were to place a thin strand of your spooled DNA on a microscope slide and stain the DNA with a blue dye,

what do you think the slide?s image might look like under the microscope? Explain your reasoning.

BACKGROUND INFORMATION: DNA Replication, PCR, and DNA Fingerprinting

The structure of the DNA double helix provides a convenient way for the cell to replicate its DNA prior to cell division:

by separation of the two DNA strands, a template is formed that allows for base-pairing of the complementary DNA

sequence of nucleotides. The enzyme that is responsible for making the phosphodiester bonds between the nucleotides on the new daughter strand is called DNA polymerase.

Scientists have learned to use DNA polymerases in the laboratory to replicate daughter strands of DNA from singlestranded DNA molecules. It is easy to tear the DNA double helix apart into single-stranded DNA in the laboratory:

since only hydrogen bonds are holding these two strands together, the strands can be separated by heat alone.

Once the DNA strands are separated, the temperature can be dropped so that the base pairs can form again, but in

the process called Polymerase Chain Reaction (PCR), ?primers? of short DNA sequences are added to the cooling

DNA to bind to specific sites of the DNA molecule where the sequence is complementary sequences of the primers.

By designing these primer DNA sequences to anneal to DNA in a region of the chromosome flanking a gene or sequence that the scientists are interested in, they can direct the DNA polymerase to replicate that specific region.

In DNA fingerprinting experiments, the primers are designed to flank a region of the chromosome where the DNA has

a variable number of tandemly-repeating sequences. As described in your textbook, these blocks of DNA, called

Short Tandem Repeats (STRs), have a varying number of repeated sequences in different people and can be used

like blood typing in forensics and paternity testing.

DNA Replication, PCR, and DNA Fingerprinting Background Questions

To better understand how the structure of DNA provides a template for copying a daughter strand by DNA poly6

merase, you may want to refresh your memory by reading about these topics in your textbook (Chapter 7). The

questions below will guide you through some relevant animations.

1. To better understand how DNA polymerase can replicate DNA in the cell, go to watch the ?DNA Replication? animation at: http://highered.mcgrawhill.com/sites/0072507470/student_view0/chapter3/animation__dna_replication__quiz_1_.html. After watching this

animation, answer the following questions in your own words.

What is the location where DNA replication begins on the chromosome in the cell?

What is the enzyme responsible for making single-stranded DNA available for replication?

Describe how the enzyme primase initiates DNA replication by DNA polymerase. Why is this primer required for replication?

Briefly describe why Okazaki fragments are made on the lagging strand of DNA during replication.

2. To better understand how DNA polymerase can be used by scientists to replicate DNA in the test tube, go to watch

the ?Polymerase Chain Reaction? animation at: http://www.dnalc.org/resources/animations/pcr.html After watching

this animation, answer the following questions in your own words.

Describe how the DNA polymerase enzyme (?Taq polymerase?) is directed to the region of interest on the

chromosome.

Describe what happens to the DNA, primers, and DNA polymerase (?Taq polymerase?) at these 3 temperature settings on the PCR thermal cycle:

94 ? 96oC

50 ? 65oC

72oC

Are the ?upstream? and ?downstream? primers the same sequence? Explain why or why not.

NOTE: The ?upstream? and ?downstream? primers are more often referred to as the ?forward? and ?reverse? primers.

What special DNA fragment appears after the 3rd thermal cycle of PCR? What defines what its length is?

NOTE: This DNA fragment is referred to as the ?amplicon?.

View the ?PCR Amplification Graph?. How many copies of DNA are made after:

10 thermal cycles

20 thermal cycles

30 thermal cycles

3. To better understand how PCR-amplified DNA can be analyzed by scientists, go to watch the ?Gel Electrophoresis?

animation at: http://www.dnalc.org/resources/animations/gelelectrophoresis.html After watching this animation, answer the following questions in your own words.

What is used to make the ?gel? for electrophoresis separations of DNA molecules?

What purposes does a ?tracking dye? play in electrophoresis?

What property of DNA makes it move in an electric field?

What property of DNA makes it separate by size in an agarose gel?

DNA is invisible to the naked eye. Describe how DNA bands can be made visible on an agarose gel.

4. To better understand how short tandem repeats (STRs) of your chromosomes can be in DNA fingerprinting go to

watch the ?DNA Variations? animation at: http://www.dnalc.org/view/15981-DNA-variations.html After watching this

animation, answer the following questions in your own words.

Describe what a repeat polymorphism is.

Explain why you have pairs of repeat polymorphisms in your chromosomes. How did you inherit these

pairs?

Explain the differences between VNTR and STR polymorphisms.

Paper Protocol: DNA Replication, PCR, and DNA Fingerprinting

Unfortunately, we will not be able to perform a real PCR experiment in this lab activity, but we can do a mock PCR experiment on paper. Follow the instructions below and answer the corresponding questions.

In this protocol, we will model a paternity test for a baby, using STRs.

In this scenario, a baby is born and there are two men who are being tested as the father. Although a full DNA fingerprint would entail looking at 13 or more STRs, we will focus our attention on just one: a trinucleotide repeat

-CAT- found on Chromosome 1. A forward and a reverse primer has been designed to flank this STR site, and is

used to PCR amplify this site from DNA samples from the baby, the mother, and the two prospective fathers. The amplicons are loaded on an agarose gel and electrophoresis is run to separate the amplicons by size. The baby?s amplicon sizes must be matched by one from the mother and one from the father.

The relevant DNA sequences can be found in the pages that follow. Notice that there are two DNA sequences for

each person, so each person will end up with two different amplicons to be separated for analysis by gel electrophoresis. There are also two primer sequences that you will use to determine where the amplicon sequences begin and end. To help you to follow the protocols below, I will show you how the steps work with the baby?s DNA.

STEP ONE: PCR AMPLIFICATION

Cut out the red-colored forward primers and blue-colored reverse primers.

Starting with the reverse primer, find its complementary sequence on each of the baby?s chromosome sequences. This would be at the coolest temperature of the thermal cycle (between 50 ? 65oC). See below

what this would look like for the baby?s DNA.

Now, add base pairs to the first strand of DNA synthesis, matching an A for every T, a C for every G. The

base pairs should fill all of the boxes provided to the right of the template strand. See below what this would

look like for the baby?s DNA.

baby's DNA sequence

baby's primed DNA sequence

primed with the reverse

primer

elongated by DNA polymerase

template

strand

(5'-end)

(3' end)

(5'-end)

(3' end)

second chromosome

template

second chromosome

template

first chromosome

template

strand

first chromosome

strand

(5'-end)

(3' end)

(5'-end)

(3' end)

A T

G C

A T

G C

A T

G C

A T

A (3' end)

A T

G C

A T

G C

A T

A (5'-end)

A T

A (5'-end)

A (3' end)

A T

G C

A T

G C

C G

A (5'-end)

(3' end)

(5'-end)

(3' end)

(5'-end)

(3' end)

STEP ONE: PCR AMPLIFICATION (continued)

Now, melt the two stands of DNA in order to amplify the 1st PCR strand, this time using the forward primers.

So, the temperature of the thermal cycle will lower to allow for annealing of the forward primer: find the DNA

sequences that are complementary to the forward primer and anneal the forward primer to its complementary sequence. See below what this would look like for the baby?s DNA.

As before, the temperature raises to 72o C to allow for the Taq DNA polymerase to make the 2nd strand of

DNA. Your job is to write in the correct base pairs to the left of the 1st strand sequence, going all the way to

the end of the DNA sequence. See below what this would look like for the baby?s DNA.

baby's 1st

strand

baby's 1st strand

primed with the forward

primer

first chromosome

second chromosome

elongated from the forward

primer

first chromosome

second chromosome

template

strand

(3' end)

(5'-end)

template

(5'-end)

T A

A T

G C

(3' end)

C G

(3' end)

(5'-end)

A T

G C

A T

G C

A T

(5'-end)

A T

G C

A T

G C

(5'-end)

(3' end)

(5'-end)

(3' end)

(5'-end)

STEP ONE: PCR AMPLIFICATION (continued)

Now, repeat the above steps, amplifying the 2nd strand with the reverse primer. Notice that this will give you

an amplicon that begins and ends with the two primer sequences. The baby?s DNA amplicons should look

like this:

baby's amplified DNA (3rd round of amplification)

first chromosome

second chromosome

(5'-end)

(3' end)

T A

A T

G C

C G

A T

T A

A T

T A

A T

T A

C G

A T

T A

A T

C G

G C

A T

G C

A T

C G

A T

(3' end)

(5'-end)

A T

G C

A T

G C

(3' end)

(5'-end)

-CAT- repeats

STEP ONE: PCR AMPLIFICATION (continued)

Now, repeat the PCR amplification steps used above for the baby?s DNA, this time amplifying the sample

DNA from the mother and the two prospective fathers. Be sure to keep track of whose DNA is whose ? perhaps by color-coding the amplicons as you create them.

Fill out the following table, indicating the number of STR repeats that you found for each of the sample

DNAs

Sample DNA

Number of STR repeats in amplicon

The baby?s

4, 6

The mother?s

The first prospective father

The second prospective father

STEP TWO: GEL ELECTROPHORESIS

Now that you have prepared amplicons from the DNA samples, you can separate them by gel electrophoresis by size. The results for the baby?s amplicons is shown below. From your data in your PCR amplicon table (above), enter the positions of the amplicons from the DNA samples of the mother and the fathers in the

gel below.

NOTE: You may hand-draw the DNA bands into this gel and paste a photo of your results here. Alternatively, you may enter the DNA bands into a gel that I have uploaded in a Microsoft Excel format on Blackboard. You may cut and paste your results into that file and paste it here.

From your gel results, which of the two men have you identified as the baby? father?

STR alleles for DNA fingerprinting by PCR

mother's DNA sequence

baby's DNA sequence

Primer sequences

first chromosome

second chromosome

first chromo

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