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1) Where is DNA found in a cell, and how do we extract it?

2) If we want to run a DNA fingerprint, how (and why) do we cut the DNA, and how does electrophoresis separate the DNA to make this pattern?

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Elin Hessel
Elin HesselLv2
28 Sep 2019
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Related questions

NA fingerprinting uses a process called gel electrophoresis toseparate the fragments of DNA. Once the DNA fragments are sorted,the pattern of bands can be analyzed.

1)Gel Electrophoresis Procedure The smaller DNA fragments startto move away from the wells and the larger DNA fragments remaincloser to the wells.

2)An electric current is passed through the gel.

3) DNA fragments are treated with a dye.

4)A restriction endonuclease is added to the DNA.

5)Using micropipettes, the DNA samples are added to thewells.

6)DNA fingerprint is produced.

7)DNA fragments are produced.

The order in which a DNA fingerprint is produced using gelelectrophoresis is:___,____,___,____,____,____,____,____

Background info:

You have identified an organism with the smallest known genome (sequence below). You isolate the DNA and digest it with EcoR1

EcoRI --- 5' G ↓AATTC 3'

5' ACGACGTATTAGAATTCTTATCCGCCGCCGGAATTCTCATCA 3'

3’ TGCTGCATAATCTTAAGAATAGGCGGCGGCCTTAAGAGTAGT 5’

Look for palindromic site, specific for restriction enzyme, each strand of the DNA is “cut” (phosphodiester bond of backbone is cleaved) at the same place in the sequence, this will produce sticky ends

The key steps in the experiment:

1. Extract DNA from organism (how would you do this, can you separate protein and nucleic acids, what about DNA from RNA)

2. Incubate DNA with restriction enzyme (what is you used the wrong enzyme? What if you accidentally boiled your sample?

3. Use gel electrophoresis to separate out DNA fragments by size (need a gel, need an electrical current, buffer)

4. Use Ethidium Bromide (EtBr) either in the gel or as a stain afterwards (this will allow for visualization of the DNA, non-specific, ring structure intercalates with bases, excited by UV light, emits orange light)

5. Include a ladder/controls – (you need ladder or something of known size if you want to estimate the size of the fragments)

6. Visualize with UV light source and imager

7. Important – maybe not as key - Use loading dye when loading DNA buffer (the buffer helps the DNA settle into the well, dye lets you approximate where the DNA should be in the gel as it is running)

______________________________________________________________________________________

Here is the question I would like to know:

1) What would happen if we changed something in the experiment?

A) What would happen if we changed step 1?

b) What would happen if we changed step 2?

c) What would happen if we changed step 3?

d) What would happen if we changed step 4?
e) What would happen if we changed step 5?

f) What would happen if we changed step 6?

g) What would happen if we changed step 7 (if anything) ?

#1. DNA testing can be done using which methods:
A. Restriction Fragment Length Polymorphism
B. Short Tandem Repeats
C. Mitochondrial sequencing
D. All of the above
E. A and C

#2. Why is protein sequencing not helpful in identifiying anindividual?

#3. PCR is a very complex process to mimic. Too much DNA and the"washing machine" is too full resulting in poor amplification. Whatsteps can we take to ensure we don't get too much DNA into thereaction (or in other words, how do we prevent too many clothesgetting in the washer)?

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