Genetics Lab Exam 2

Now look at the upper sequence of bases and compare it to the lower. Do you notice any grouping of bases that when read toward the right on the upper strand and read to the left on the bottom strand are exactly the same?

CTTAAG

Restriction site

are locations on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes

How many base pairs are there to the right of the cut?

10

How many steps does PCR have?

3; denature, anneal, and extend

How many base pairs are there to the left of the cut?

4

If the GAATTC palindrome is repeated four times on the same piece of linear DNA, and the restriction enzyme that recognizes that base sequence is present, how many DNA fragments will be produced?

5

Compare the bases in the upper DNA strand to those in the lower strand. Describe any relationship you can see.

A always pairs with T and C always pairs with G

What does this tell you about the status of your food?

A band indicates that the food may be GMO positive, and the absence of a band indicates the food may be GMO negative.

Annealing step of PCR

DNA is cooled to 59 degrees celcius to allow the primers to locate and bind to the DNA; they will anneal much more quickly than the long template DNA strands at this temperature

Explain why DNA fragments separate according to size in an electrophoresis gel.

DNA is negatively chaged and is repelled by the negative electrode (cathode) and attracted by the positive electrode (anode) when an electric current is applied across the gel. It separates because different lengths of DNA move through the gel matrix at different rates. Longer fragments move more slowly than shorter fragments.

What molecules are present in the cell that might interfere with DNA extraction?

Enzymes, such as DNases, may degreade DNA. Metal ions act as cofactors and coenzymes for enzymes that degrade DNA. Cellulose plant cell walls may act as a barrier to DNA extraction.

How could you describe the fragment size in reference to the number of base pairs in the fragment?

Fragment 1 is a 4-base pair fragment. Fragment 2 is a 10-base pair fragment.

A restriction enzyme cuts between G and A in the palindromic sequence

GAATTC

Why did you resolve your PCR products by electrophoresis?

Gel electrophoresis separates DNA molecules based on charge and size. After the bands are separated the gel is stained to visualize the band pattern. We can calculate the size of the DNA molecules, in base pairs, in each band.

Many foods containing GM crops are highly processed. Can you suggest how DNA from whole plants may differ from that extracted from processed foods, e.g. corn chips, cornmeal, etc.?

High temperatures or physical manipulation of the plant tissue during processing may destroy or fragment DNA.

What other information do you need to confirm the GMO status of your sample?

If there was a band, we need to determine that there was not contamination of the samples to ensure the result is not a false positive. If there was no band, we need to confirm that the DNA extracted from the sample and that the PCR reaction was functioning properly to assume there was not a false negative.

Why was the non-GMO food control prepared prior to your test food sample?

In the grinding process, airbourne particles can travel through the air and contaminate samples of non-GMO foods. Also, a mortar and pestle that is not properly washed can transfer minute sample. PCR only needs one molecule of DNA to make an amplified product.

If the DNA in tube L becomes fragmented at the conclusion of the reaction, what can you conclude?

Most likely a restriction enzyme was inadvertently added to the L tube. This could have happened by accidently adding an enzyme to the tube, or by not changing pipet tips, which could result in enzyme being carried over between tubes.

Did your test food generate a 200 bp band with GMO primer?

No

Is the DNA visible?

No

What is missing in that tube that leads you to that decision?

No restriction enzymes are added to that tube, thus no digestion will occur, and no fragments will be produced. This tube, L, is the control tube.

Counting the number of base pairs, is the right fragment the same size as the left fragment?

No, it is larger.

Is there any visible change to the DNA after adding restriction enzymes?

No. The DNA still appears colorless.

Why are you perfoming 2 PCR reactions on each DNA sample?

One reaction is a control to show we extracted plant DNA using primers to a universal plant DNA sequence. The second reaction is to identify the GMO target sequence.

In what organelles is plant DNA located?

Plant DNA is not only found in the nucleus, it is also found in the mitochondria and chloroplasts. Plants and other autotrophic organisms are the only organisms with chloroplasts. Plant DNA is more difficult to obtain intact because the cell wall must be destroyed.

If the GAATTC palindrome repeats are randomly spaced along the DNA strand, then what can you say about the size of the fragments that will be produced when the DNA is digested with a restriction enzyme that recognizes that sequence?

Random sized fragments will be produced.

Why do you expect this difference?

Restriction enzymes digest DNA at specific sites. If lambda DNA contains restriction sites for the enzyme PsfI, then the DNA should be cut into fragments. With no added enzyme, no digestion should occur.

What was your test food?

Smartfood white cheddar popcorn and sunchips

What chemicals and molecules are needed for PCR, and what is the function of each componet?

Taq DNA polymerase, Deoxynucleotide triphosphates, primers, buffers, and cofactors

In which tube do you expect no changes to occur-- that is, no DNA fragments produced?

The DNA in the uncut lambda DNA tube, which gets no enzyme, should remain intact as a single band.

Describe the appearance of the DNA in solution.

The DNA is colorless

Compare the P tube to the L tube; what do you expect to happen in the P tube compared to the L tube?

The P tube contains the restriction enzyme PStI. There should be an enzymatic digestion that occurs in that tube, which results in the production of restriction fragments. The L tube only contains DNA and no enzyme and so should not produce any restriction fragments.

Extension step of PCR

The temperature is increased to 72 degrees celcius, the optimal temperature for the DNA polymerase to function. DNA polymerase adds nucleotides one at a time at the 3' end of the primer to create a complementary copy of the original DNA template.

How can you test a food to find out if it contains material derived from a genetically modified organism (GMO)?

There are two methods to test for foods containing GMOs. The ELISA test is used to see if particular proteins are in a sample. PCR is used to amplify regions of GMO genomes.

Describe any pattern you might see in the upper sequence of the bases.

There is no specific type of pattern associated with the upper sequence of the bases

Why do you also perform analysis on food that is known to be non-GMO control?

To make sure the samples have not been contaminated. It is also used as a comparison to show how a non-GMO banding pattern should look.

Why do you need a molecular mass ruler alongside your samples?

We need a molecular mass ruler to calculate the size of each of our bands. We know exactly how many bands are in the ruler and the size of each of those bands. We can graph the size of the bands against the distance they moved in the gel to create a standard curve. We can then measure the distance our PCR product bands moved in the gel and use our standard curve to calculate the sizes of the product bands.

What is the purpose of the GMO positive control DNA?

We want to make sure our PCR reaction worked; if the positive control produces a positive result, but we do not get a band in our test sample, the test is most likely non-GMO. If we do not get the 200 base pair band in the positive control, we can assume the PCR reaction did not work.

PCR (polymerase chain reaction)

a laboratory technique used to make multiple copies of a segment of DNA; very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules.

Palindrome Sequence

a nucleic acid sequence (DNA or RNA) that is the same whether read 5' (five-prime) to 3' (three prime) on one strand or 5' to 3' on the complementary strand with which it forms a double helix.

Taq DNA polymerase

a polymerase that is not sensitive to heat. It links the deoxynucleotide triphosphates to make a DNA strand that is complementary to the template

"Sticky End"

a short length of unpaired bases at the DNA site some restriction enzymes leave where they cut

Buffers and cofactors

needed to make the reaction take place at an optimal rate

Primers

short sequences of DNA that serve as beginnings of newly synthesized DNA

Denaturing Step of PCR

the DNA template is heated to 94 degrees celcius to separate the double-stranded DNA into two single strand.

Deoxynucleotide triphosphates

the basic units that are connected to make the complementary strand