Group No: Section: PSY5B

Name: Uy, Bryan Gunther T. Score:

Exercise 10

The Tools of DNA Technology

DNA Extraction

DNA is extracted from human cells for a variety of reasons. With a pure sample of DNA you can
test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer. Check
this website and enter the virtual laboratory to perform a cheek swab and extract DNA from human cells.
http://learn.genetics.utah.edu/units/biotech/extraction/

Gel Electrophoresis

Scientists can identify DNA segments by measuring how long they are using a technique called
gel electrophoresis. Visit this website and run your own gel electrophoresis experiment to sort and
analyze DNA strands in the biotechniques virtual laboratory.
http://learn.genetics.utah.edu/units/biotech/gel/

Restriction Fragment Length Polymorphisms Analysis

Scientists can compare the DNA sequences of different individuals by exploiting the presence of
restriction fragment length polymorphisms (RFLPs). The number of restriction fragments and their sizes
reflect the specific sequence of nucleotides in your DNA.

In this exercise, concepts and analyses using RFLP will be demonstrated. Such activities will
show how variations among closely related organisms can be facilitated and demonstrate the diagnosis of
certain human genetic diseases. (Note: The human genome contains about three billion base pairs. For
classroom purposes, the exercise will utilize small sections of DNA to simulate how RFLPs are used to
detect genes causing certain disorders.)

I. Analyses of RFLP Using a Hypothetical DNA sequences

Figure 1 refers to two DNA strands obtained from identical regions of a hypothetical disease-
causing gene in a particular chromosome from two different individuals. Each strand is 500 base pairs
long.

Table 1 below lists some of the more common restriction enzymes and the restriction sites they
recognize in the DNA. Note that these restriction enzymes cleave the phosphodiester bonds in the double
stranded DNA, producing either sticky ends or blunt ends.
 Table 1. Some restriction enzymes and their restriction sites.

A. Cutting DNA into Fragments

Locate the restriction sites for EcoRI and HindIII in individual 1, which the enzymes will cut to
produce several fragments. Enclose these restriction sites with boxes. Now refer to individual 2. Note that
the disease-causing gene you are searching for is linked to a mutation that destroys a restriction enzyme
cleavage site. Therefore, individual 2 will lose at least one particular restriction site since the restriction
enzyme no longer recognizes the site where the mutation took place, and the number of fragments as well
as the size of fragments that will produced will differ from those in individual 1.

B. Separating DNA Fragments by Size: Gel Electrophoresis

The DNA fragments are normally separated according to size by electrophoresis. In principle, the
DNA fragments that are loaded in a gel will separate as they migrate down the gel as an electric current is
allowed to pass through it. Determine the number of fragments produced by restriction digest for
individuals 1 and 2 as well as the approximate length of each fragment (Refer to the table below). If the
gel contains ethidium bromide (EtBr) or is submerged to this chemical, the EtBr will bind to the DNA
fragments. Subsequently, once the gel is placed under UV light, the DNA fragments will be visualized
asdistinct bands because EtBr fluoresces under UV light.

The box below represents the gel used for electrophoresis, and the first lane represents DNA fragments
with specific lengths in base pairs (bp) that are used as molecular weight markers (MW). Indicate the
bands and their approximate lengths for individual 1 (lane 2) and individual 2 (lane 3)

Individual 1 Individual 2

Fragment Length of Fragment Fragment Length of Fragment

1 53 bp 1 53 bp
2 223 bp 2 170bp
3 345 bp
4 106bp 4 228 bp
C. Transferring DNA fragments: Southern blotting

Sometimes, it may be necessary to identify if specific sequences are found in the DNA fragments.
Prior to detection of these specific segments, the DNA fragments from the gel must first be transferred to
a nitrocellulose membrane through Southern blotting. During transfer, however, the relative positions of
the bands in the gel are not changed, so the profile of the DNA fragments in the gel is the same as that in
nitrocellulose membrane.

D. Probing the DNA: Hybridization

Figure 2 represents a probethat will be used to bind to DNA and visualize it using Southern Blot
Analysis. The letters in boldface represent radioactively labeled bases that are incorporated into the
probe. Refer to figure 1. Match the base pair sequence of the probe to its complementary DNA sequence
for each fragment that is supposedly transferred into the nitrocellulose membrane. Does this by encircling
the sequence found in each segment that matches the probe. Note that sometimes, not all fragments will
be hybridized to the probes.

5’ AATTTCTATT 3’
3' TTAAAGATAA 5'

Figure 2: DNA probe

E. Visualizing the RFLP: Autoradiography

The final step is to visualize where the probe is hybridized. In the lab, and X-ray film is placed
on the membrane. A dark band appears on the developed film directly over the region where the
radioactive probe hybridized to RFLP. This process is known as autoradiography, and the developed
filmis called autoradiograph. Write below how the new profiles for individual 1 and 2 will appear after
Southern Blot Analysis.
Ind. 1 5' ACCTGCTGAT GGAATTTCTA TTAGGATTCT TTAGGCCTAG TAAGCTCGAC 50

Ind. 2 5' ACCTGCTGAT GGAATTTCTA TTAGGATTCT TTAGGCCTAG TAAGCTCGAC 50

Ind. 1 TCGAATTCGA GCTTAGCCCA GTCAACTTGC CTGGGAATAC TTTTAAATCT 100

Ind. 2 TCGAATTCGA GCTTAGCCCA GTCAACTTGC CTGGGAATAC TTTTAAATCT 100

Ind. 1 AATTTCTATT CTCTCTGAGG GAACCTTGAC AGAGCCCTAC CACACAGGTT 150

Ind. 2 AATTTCTATT CTCTCTGAGG GAACCTTGAC AGAGCCCTAC CACACAGGTT 150

Ind. 1 GGCGATTTAA TTTCTATTCG CCCCGGAAAT TGTGCACAGC TTTGCAAACT 200

Ind. 2 GGCGATTTAA TTTCTATTCG CCCCGGAAAT TGTGCACAGC TTTGCAAACT 200

Ind. 1 TCCCGGGCAA CATGGTAACT TCAAGCTTAC GAATTTCTAT TCCCAATGAC 250

Ind. 2 TCCCGGGCAA CATGGTAACT TCAAGCTTAC GAATTTCTAT TCCCAATGAC 250

Ind. 1 GGGGCCTTCA GCGAAATTCA AATTTCTATT GGACACTATA TCTGGCTAAA 300

Ind. 2 GGGGCCTTCA GCGAAATTCA AATTTCTATT GGACACTATA TCTGGCTAAA 300

Ind. 1 ACACTGGACC CTCTGAGATC CGGCCTCCCT CGAATCCTTC GCCTGAATTC 350

Ind. 2 ACACTGGACC CTCTGAGATC CGGCCTCCCT CGAATCCTTC GCCTGAAGTC 350

Ind. 1 ACTTTTCCCG ACTGGCTTGA ATTCGGCCAA GTACATTAAT TTCTATTAGT 400

Ind. 2 ACTTTTCCCG ACTGGCTTGA ATTCGGCCAA GTACATTAAT TTCTATTAGT 400

Ind. 1 CAGAACTCGA ATTCTCCGGA CGCTTGATGC GAGTTACCCC CGGCTTTTCC 450

Ind. 2 CAGAACTCGA ATTCTCCGGA CGCTTGATGC GAGTTACCCC CGGCTTTTCC 450

Ind. 1 AAGCTTTTCG TTCCGTGTCC AACTTGATTA CGCGTTGAAA CGGTCGCTAA 3' 500

Ind. 2 AAGCTTTTCG TTCCGTGTCC AACTTGATTA CGCGTTGAAA CGGTCGCTAA 3' 500

Figure 1: DNA segments obtained from identical regions of a hypothetical disease-causing gene in a
particular chromosome from two different individuals.
 II. Application of RFLP: Identification of Acanthamoeba Isolates Using the Small Subunit (SSU)
Ribosomal DNA Gene

Acanthamoeba is a free-living soil amoeba that can produce a resistant cyst. Some members of
this genus can cause granulomatous amoebic encephalitis (GAE) in humans.

The small subunit (SSU) ribosomal DNA gene of six Acanthamoeba isolates were isolated and
amplified through polymerase chain reaction (PCR) after which the gene was subjected to restriction
enzymes using Hinf I and Taq I. The profiles below show the DNA fragments without being subjected to
Southern Blot Analysis and instead are visualized using ethidium bromide. A 100-bp ladder is used to
serve as marker.

1. Which isolates are most similar based on the Hinf I restriction digest?
Isolates 1,4 and 6

2. Which isolates are most similar based on the Taq I restriction digest?
Isolates 2, 3 and 5

3. How do differences in banding banding patterns translate to genetic variations among the isolates?
The genetic variation should be referred to the marker, which is the 100 bp marker. Among the
isolates the bands that are closest to the marker are translated are most similar to the restriction
enzyme
 III. Application of RFLP: Southern Blot Analysis of Duchene Muscular Dystrophy (DMD)

Duchene Muscular Dystrophy (DMD) is a sex-linked recessive trait involving lingering or

chronic muscle weakness. In advanced cases, respiratory muscles may also be affected, thus
compromising normal respiration. It is also accompanied by weakened pumping of the heart.

The pedigree below of a certain family with a history of DMD is accompanied by an

autoradiograph of a Southern blot. The DNA from the DMD locus from each member was digested
usingHindIII.

Which individual(s) is/are affected with DMD? Justify your answer. Can you identify which individual
(s) is/are probable carriers of the disease? Why do you think so?

The individual that is affected with DMD is the Individual 6. It is individual with hetero (half band) or
homozygous (no band) recessive for the trait. Since Individual 1 and 5 are carrier. This where DNA
polymorphism is displayed. Half band is carrier, and no bands is affected with DMD, regardless of the
gender. Since it is a recessive trait in the RFLP, it shows that the missing band displays trait while half band
is carrier which is 1 out of 6.

IV. Application of RFLP: Mapping the Restriction Sites

A. A 12-kb circular DNA from a certain bacterium is cut twice by both Eco RI and Hind III. EcoRI
yields fragments of 7 kb and 5 kb, and Hind III yields fragments of 8 and 4 kb. Eco RI-A is cut by Hind
III to yield two fragments: 3 kb and 4 kb. Eco RI-B is cut by Hind III to yield two fragments: 1 kb and 4
kb. Draw a diagram of the circular DNA with all 4 restriction sites.
 ECORI 8 kb

HIND III

7 kb 12-kb 4 kb

1k ECORI b
b
4 kb

4 kb
HIND III

ECOR
I 5kb

B. A linear fragment of DNA was cleaved with the individual restriction enzymes Hind III and Sma I,
and then with a combination of the 2 enzymes. The results were the following:
Hind III alone Sma I alone Hind III + Sma I
2.5 kb 2.0 kb 2.5 kb
5.0 kb 5.5 kb 3.0 kb
2.0 kb
Draw the restriction map.
2.0 Hind 3.0 Sma I 2.0

V. Applications of RFLP: Medical Diagnostics

In search for the gene for the genetic disease using restriction fragment polymorphisms, DNA was
isolated from five people with the disease, five without the disease, and 2 patients with unknown
condition. The DNAs were treated with a restriction endonuclease and the digested fragments separated
by agarose electrophoresis. Based on the results given below, what is your diagnosis for each patient with
unknown condition? Justify your answer.

- Unknown 1 has the disease. Unique bands among people with disease are present at 300 and 320 bp from
fragments 1 to 5 and as seen in Unknown 1, those unique bands are present thus, the unknown person has the
disease.

- Unknown 2 is a normal heterzygous. This is because there is a presence of both unique bands from people
without disease and people with disease. Meaning there is a normal copy the disease and an abnormal copy of
it. As seen in fragments 4 and 5 of people without disease and people with disease are aligned with the
fragments of the Unknown 2 patient, thus becoming normal heterozygous.