the biotechnology education company p d a t e d r e v i s e d a d n the biotechnology education...

Updated

Revised

and

The Biotechnology Education Company ®

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

EVT 001227K

EDVO-Kit #

314In Search of the Cancer Gene

Storage: See Page 3 for specifi c storage instructions

EXPERIMENT OBJECTIVE:

In this experiment, students will gain an understanding of the effect of mutations in the p53 tumor suppressor

gene and its role in familial cancers.

This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.

In Search of the Cancer Gene

EVT 001227K

2

xxx314EDVO-Kit #

EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

Page

Experiment Components 3Experiment Requirements 4Background Information 5

Experiment Procedures Experiment Overview and General Instructions 12 Module One: Construction of a Family Pedigree 13 Module Two: Separation of DNA Fragments 14 Module Three: Analysis of Autorads to Search for p53 Mutagens 17 Study Questions 18 Instructor's Guidelines Notes to the Instructor 19 Experiment Results and Analysis 23 Study Questions and Answers 25

Appendices

A Agarose Gel Preparation For DNA Staining with InstaStain® Ethidium Bromide 28

B Quantity Preparations for Agarose Gel Electrophoresis 29 C Staining and Visualization of DNA with InstaStain® Ethidium Bromide Cards 30

Material Safety Data Sheets 31

Table of Contents


EVT 001227K

3

EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com

FAX: (301) 340-0582 • email: [email protected]

314EDVO-Kit #

Experiment Components

All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor admin-istered to or consumed by humans or animals.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experi-ment components are de-rived from human sources.

Ready-to-Load Samples for Electrophoresis

A Standard DNA Fragments B Control DNA C Patient Peripheral blood DNA D Patient Breast Tumor DNA E Patient Normal Breast Tissue DNA

Reagents & Supplies

• X-ray simulated p53 hot spot sequences • UltraSpec-Agarose™ powder • Concentrated electrophoresis buffer • InstaStain® Ethidium Bromide

This experiment contains reagents for 6 groups.

This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.

Components & Requirements

Before use, check all volumes of Standard DNA fragments and DNA samples for electrophoresis. Evaporation may have caused samples to become more concentrated.

If needed, tap tubes or centrifuge, then add distilled water to slightly above the 1.0 cm level and mix.

Storage: All DNA samples can be stored in the refrigerator

4.1

cm

ApproximateVolume

Measurements

0.5 cm tube120 µl

1.0

cm


EVT 001227K

4

xxx314EDVO-Kit #

EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Mon - Fri 9 am - 6 pm ET

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Mon - Fri9:00 am to 6:00 pm ET

FAX: (301) 340-0582Web: www.edvotek.comemail: [email protected]

Please have the following information ready:

• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date

Technical ServiceDepartment

Online Orderingnow available

Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments forbiotechnology and biology education.

Requirements

• Horizontal gel electrophoresis apparatus • D.C. power supply • Automatic micropipets with tips • Balance • Microwave, hot plate or burner • Pipet pump • 250 ml fl asks or beakers • Hot gloves • Safety goggles and disposable laboratory gloves • Distilled or deionized water • UV Transilluminator


314EDVO-Kit #

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1997, 1998, 1999, 2001, 2002, 2006 EDVOTEK, Inc., all rights reserved EVT 001227K

Backg

rou

nd

Info

rmatio

n

About Family Pedigrees

• A Circle represents a female.

• A square represents a male. • A shaded circle or square refers to a per-

son having some form of cancer.

• An open (non-shaded) square or circle represents a person who is free of cancer.

• A circle or square (either shaded or open)

with a diagonal slash through it repre-sents a person who is deceased.

In Li-Fraumeni syndrome, the pattern of cancers in family pedigrees suggest dominant inheritance. It is a genetic predisposition leading to specifi c types of cancers. Typically, the onset of cancer is at an early age, with multiple primary tumors.

When drawing a family pedigree, the following are general guidelines to the symbols used and their representations:

Female free of cancer

Male free of cancer

Female with some form of cancer

Male with some form of cancer

Female , deceased

Male, deceased

or

or


6



EDVO-Kit #B

ackg

rou

nd

Info

rmat

ion

The Role of Genes in Cancer

Many contributory factors have been identifi ed to cause the onset of cancer that include exposure to certain carcinogens in our diets and environment. Several forms of cancer have familial predisposition. These cancers appear to be linked to inherited mutation of suppressor genes, such as p53.

Hereditary SporadicGermline Mutation Somatic Mutation

Multiple Tumors

Bilateral Tumors

Early onset

Somatic Mutation

Single Tumors

Unilateral Tumors

Later onset

Normal Gene

First Somatic Mutation

Second Somatic Mutation

Figure 1: Hereditary and Sporadic Models of Gene Inactivation.

Familial cancers constitute a very small fraction of the total reported cancers and occur in dominant inherited patterns. Mutations that are directly inher-ited are referred to as germline mutations. Such mutations can be detected in familial pedigrees. A second type of mutation, known as somatic mutations, do not have direct genetic links and are acquired during the life of the individual. Patterns of typical hereditary and sporadically ac-quired nonhereditary pedigrees appear in Figure 1.

In a germline with an inherited mutation, a single somatic muta-tion within a suppressor gene will result in the inactivation of both alleles. By contrast, nor-mal inherited suppressor genes, that are free of mutations, will require two sequential mutations to initiate tumors. This model is referred to as the "Two-hit" hypothesis.

Historically, some of the fi rst genes identifi ed include the reti-noblastoma (RB) gene, Wilm's’ tumor (WTI), neurofi bromatosis type II gene and Li-Fraumeni syn-drome. In Li-Fraumeni syndrome, a notable feature in family pedi-grees, include a sarcoma patient and at least two immediate rela-

tives with other cancers before the age of 45, as well as multiple cancers in other family members. This is illustrated in fi gure 2.


314EDVO-Kit #



Backg

rou

nd

Info

rmatio

n


With the advent of molecular biology applications to medicine, gene maps and the chromosomal locations of genes are available as tools for the identifi cation of predisposition for various diseases. The procedures used to obtain such information include DNA isolation and the analysis of point mutations in hot spot areas in cancer-related genes, such as p53. Several methods of analysis for the detection of point mutations in genes include DNA sequencing.

BB.42

BR.36 SS.23BB.34

CN.2 CN.36

OS.13 LK.2

BB Bilateral breast cancerBR Breast cancerCN Brain tumorLK LeukemiaOS Osteosar comaSS Soft tissue sar coma

Figure 2: Example of a Li-Fraumeni family pedigree.

The Human Genome Project has provided information to link identifi cation of various cancers and other diseases to DNA sequenc-ing information. This information needs to be handled cautiously to assure confi dential-ity of patient genetic profi les.

The study of inherited cancers has given cancer molecular biologists the opportunity to search for genes that are critical in normal cell development and carcinogenesis. At the molecular level, cancer formation is char-acterized by alterations in both dominant oncogenes and tumor suppressor genes, such as p53. Suppressors are normal cellular proteins that are involved in limiting cell growth. By contrast, oncogenes are involved in promoting the growth of cells.

In recent years, the p53 tumor suppressor protein has become the center of many cancer biology studies. Because it appears to be of major signifi -cance, there is great impetus to study how this gene functions in normal cells compared to cancer cells. The gene for the p53 protein is located on the short arm of chromosome 17. It encodes a normal 53,000 molecular nuclear phosphoprotein. Wild type p53 functions as a cell regulator. There is now well-documented evidence that normal p53 is a sequence-specifi c DNA-bind-ing protein that is a transcriptional regulator. Upon introduction of muta-tions, p53 loses its ability to bind to DNA. By contrast, p53 that have muta-tions in specifi c hot spots promote uncontrolled cell growth and therefore function as oncogenes. For a tumor suppressor gene such as p53 to play a role in transformation in cancer, both alleles need to be altered, as shown in fi gures 1 and 2.

The p53 protein can be divided into three domains. The fi rst is the amino terminus region which contains the transcriptional activation region. The second is the central region within the protein where the majority of critical "hot spot" mutations are located. These "hot spots" are sites where muta-


8



EDVO-Kit #B

ackg

rou

nd

Info

rmat

ion


tions are detected in high frequencies. They are between exons 5 through 8 where 95% of the mutations occur. Within this region there are fi ve sub-regions where point mutations are detected in human cancers. The third region of the p53 protein is the carboxyl section, the most complex section that contains the oligomerization and nuclear localization sequences.

Examples of hot spots include codons 165 and 175 in exon 5; 196 and 213 in exon 6; 245 and 248 in exon 7; 273 and 282 in exon 8; all are within the p53 protein. Several of these mutations result in an altered p53 protein confor-mation. In turn, these changes can result in increased stability of the mutant protein and the ability to bind to the normal p53 protein and inactivate it. It is of interest to note that there are correlations between mutation and tumor tissue. One example is the mutation at amino acid 175 which is com-mon in colon carcinoma but is rarely observed in lung carcinoma. The inherited Li-Fraumeni syndrome as it has become to be known is rare. When it does occur it affects young family members and results in high mor-tality rates. Two physicians, Li and Fraumeni fi rst described the syndrome after examining death certifi cates of 648 childhood sarcomas. It was discov-ered in four families where siblings and cousins had childhood sarcomas. Further analysis showed more than 50% of the affected families had extend-ed phenotypes that included brain, breast cancers and leukemias. Cells from individuals with LFS have only a single wild type p53 allele. Examination of their p53 gene have shown correlations of the cancers to mutations in the protein as described above.

OVERVIEW OF POLYMERASE CHAIN REACTION:

PCR has two important advantages. The fi rst is sensitivity, which allows for DNA fi ngerprinting identifi cation using much smaller amounts of DNA since PCR amplifi es DNA. The second advantage is the speed of PCR analysis, which allows critical questions to be answered more quickly as compared to Southern Blot analysis.

PCR amplifi cation requires the use of a thermostable DNA polymerase, such as Taq polymerase. Purifi ed from a bacterium known as Thermus Aquaticus that inhabits hot springs, Taq polymerase is commonly used in PCR because it remains stable at near-boiling temperatures. Also included in the PCR reaction are the four deoxynucleotides (dATP, dCTP, dGTP, and dTTP) and two synthetic oligonucleotides, typically 15-30 base pairs in length, known as "primers". These components, together with the DNA to be amplifi ed, are incubated in an appropriate buffer that contains Mg2+. The primers are designed to correspond to the start and end of the DNA to be amplifi ed, known as the "target".


314EDVO-Kit #



The Exp

erimen

t


3' 5'

3' 5'

5' 3'

5' 3'

5'

5' 3' 3' 5'

5' 3'

5' 5'

Denature 94°C

5'

Extension72°C

3' 5'

Separation of 2 DNA strands

=

Primer 1 =

Primer 2 =

5' 3' 5'

Anneal 2 primers

45°C

3' 5' 5'

5' 5'

3' 5' 5'

5'

5' 3'

5'

5' 5'

5' 3'

5' 3'

5' 3'

5' 3'

5' 3'

5' 3'

5'

5' 3'

Cyc

le 1

C

ycle

2

Cyc

le 3

Target Sequence

5' 3'

5' 3'

5' 3'

Figure 3: The Polymerase Chain Reaction


10



EDVO-Kit #Th

e Ex

per

imen

t

The PCR reaction mixture (which contains the DNA polymerase, buffer, deoxynucleotides, primers, and template) is subjected to sequential heating/cooling cycles at three different temperatures (Figure 3).

• In the fi rst step, the enzyme reaction is heated to near boiling (92° - 96°C.) to denature or "melt" the DNA. This step, known as "denatur-ation" disrupts the hydrogen bonds between the two complimentary DNA strands and causes their separation.

• In the second PCR step, the mixture is cooled to a temperature that is typically in the range of 45° - 65°. In this step, known as "annealing", the primers, present in great excess to the template, bind to the sepa-rated DNA strands.

• In the third PCR step, known as "extension", the temperature is raised to an intermediate value, usually 72°C. At this temperature the Taq polymerase is maximally active and adds nucleotides to the primers to synthesize the new complimentary strands.

OVERVIEW OF DNA SEQUENCING:

During DNA sequence analysis, four separate enzymatic reactions are per-formed, one for each nucleotide. Each reaction contains DNA polymerase, the single-stranded DNA template to be sequenced and to which the synthetic DNA primer has been hybridized, the four deoxyribonucleotide tri-phosphates (dATP, dGTP, dCTP, dTTP), 32P labeled or fl uorescent nucleotide(s) are added for the detection of growing DNA fragments (Figure 4), and the appropriate buffer for in vitro DNA synthesis.

In sequencing reactions, the “G” reaction contains dideoxyGTP, the “C” reac-tion dideoxyCTP, the “A” reaction dideoxyATP, and the “T” reaction dide-oxyTTP. The dideoxynucleotide concentrations are carefully adjusted so that they are incorporated into a growing DNA strand randomly and infrequent-ly. Once a dideoxynucleotide is incorporated into the growing DNA strand, DNA synthesis is terminated. The site of the dideoxynucleotide incorpora-tion allows one to determine the position of that base. The dideoxynucleo-tide lacks a 3'-OH group on the ribose ring and as a consequence, DNA synthesis is terminated because the DNA polymerase will not add another nucleotide to the growing strand since a 3'-OH group is absolutely required for DNA chain elongation.

Since a particular reaction will contain millions of growing DNA strands, a “nested set” of fragments is obtained with each fragment being terminated at a different position corresponding to the random incorporation of the dideoxynucleotide.



314EDVO-Kit #



The Exp

erimen

t

DeducedSequence

GAGCCAGCGAGTA

FragmentSize

31302928272625242322212019

C T A G

3’

5’

Figure 4: Determining the sequence of a DNA fragment.

Figure 4 shows a “nested set” of fragments produced for a hypothetical DNA sequence. The "G" reaction contains the standard reaction mixture (dATP, dCTP, dGTP, dTTP, the DNA polymerase, the appropriate buffer for DNA synthesis, 32P labeled or fl uorescent tagged nucleotide(s) and a small amount of dideoxyGTP (ddGTP). The ddGTP (dideoxyGTP) random and infrequent incorporation will produce a “nested set” of fragments which terminate with a ddGTP. The “nested set” is complimentary to the sequence. Similar “nested sets” are produced in the separate “A”, “T”, and “C” reac-tions. For example, the “A” “nested set” would terminate with a ddATP.

It should be apparent that together the “G, A, T, C” “nested sets” con-tain fragments ranging in size successively from 19 to 31 nucleotides for the hypothetical sequence in Figure 3. In the fi gure the “G” reaction contains fragments of 21, 23, 25, 29 and 31 nucleotides in length. The fi rst eighteen nucleotides are contained in the synthetic DNA sequenc-ing primer which are not shown. The rest are added during de novo synthesis by the DNA polymerase.

The reaction products from the G, A, T, and C reactions are separated by electro-phoresis using a thin and long vertical polyacrylamide gel. DNA sequencing gels

resolve fragments which differ in size by a single nucleotide. After elec-trophoretic separation is complete, the sequence is determined by either radioactivity or fl uorescence. If radioactivity was used, autoradiography is performed. The polyacrylamide gel is placed into direct contact with a sheet of x-ray fi lm. Since the DNA fragments are radioactively labeled with 32P, their position can be detected by a dark exposure band on the sheet of x-ray fi lm. Recent advances have also made it possible to automate DNA sequenc-ing and to avoid the use of isotopic 32P.



12



EDVO-Kit #Th

e Ex

per

imen

t

EXPERIMENT OBJECTIVE:

In this experiment, you will construct a family pedigree that is suspected to have the classic Li-Fraumeni syndrome. DNA samples which have been enzy-matically digested will be separated by electrophoresis on an agarose gel as an independent diagnostic test. DNA sequencing x-rays simulating p53 hot spot sequences will also be examined and mutations will be identifi ed.

LABORATORY SAFETY

1. Gloves and goggles should be worn routinely as good laboratory prac-tice.

2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the laboratory.

5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

LABORATORY NOTEBOOK RECORDINGS:

Address and record the following in your laboratory notebook or on a sepa-rate worksheet.

Before starting the Experiment:

• Write a hypothesis that refl ects the experiment. • Predict experimental outcomes.

During the Experiment:

• Record (draw) your observations, or photograph the results.

Following the Experiment:

• Formulate an explanation from the results. • Determine what could be changed in the experiment if the experi-

ment were repeated. • Write a hypothesis that would refl ect this change.

Experiment Overview and General Instructions


314EDVO-Kit #



The Exp

erimen

t

Module One: Construction of a Family Pedigree

A fi rst step in the search and assignment of Li-Fraumeni syndrome is to es-tablish the family pedigree of the patient.

The fi rst part of the experiment is based on the information made avail-able as part of a diagnosis by the family physician and the oncologist. The pedigree information that you will develop is for a young woman who is suspected to have the Li-Fraumeni syndrome.

INFORMATION FOR DEVELOPING THE FAMILY PEDIGREE:

Upon monthly breast self-examination, Valerie Brown, age 36, found a small irregular mass. She was concerned because she knew that her mother had a mastectomy when she was in her late thirties. Valerie made an appointment with her physician, who referred her to a specialist at a local cancer center, where she was diagnosed as having breast cancer. As part of the medical work-up, the oncologist had inquired about her family history of cancer. Upon consultation with her mother, Valerie learned that her father and his family appeared to be free of cancer. However, in Valerie's mother's family, several cases of cancer have occurred.

With the information given below, chart the family pedigree.

• Her mother, Diane, was diagnosed and treated for breast cancer at the age of 39.

• Valerie did not know that Diane had a sister, Mabel, who died at age 2

of a brain tumor.

• Diane's brother, James (age 40), underwent surgery, followed by chemo-therapy for colon cancer.

• Her maternal grandmother, Elsie, died at age 42 from bilateral breast cancer.

• Her maternal grandfather, Elmer, was free of cancer and is 88 years old.

• Her maternal cousin, Patrick (son of James), died of brain cancer at 14.

• Her cousin (Patrick's sister), Jane, was diagnosed with childhood leuke-mia and subsequently died at age 2 .

• Patrick's two other brothers, Richard (age 28) and Curtis (age 30), are in good health and free of cancer.

• Valerie's sister, Nancy (age 38), is free of cancer.


14



EDVO-Kit #Th

e Ex

per

imen

t

Module Two: Separation of DNA Fragments

The familial pedigree in Module 1 strongly suggests Li-Fraumeni syndrome. In such a case, a secondary diagnostic test is normally conducted. In this scenario, Valerie provides a sample of blood and tumor biopsy tissue to con-duct DNA analysis for the p53 gene. Normally the procedure is to amplify the gene using polymerase chain reaction. This is followed by one of several methods to detect the presence of a point mutation at the hot spots.

In this simulation experiment, Valerie's DNA has already been digested with a restriction enzyme that recognizes the mutant sequence at the simulated hot spot site at nucleotide 165 which is also the palindrome CAGCTG for the restriction enzyme. This restriction enzyme was used as a probe to cut the simulated amplifi ed gene for Valerie’s DNA sample, together with a normal control and a set of standard DNA marker fragments. Digestion of the normal amplifi ed DNA will give a characteristic "control" DNA fragment banding pattern. The DNA obtained from blood lymphocytes will give an altered band pattern representing one normal allele and the second which is the mutant. The DNA analysis from the tumor tissue will show only the pattern for the tumor allele. The predigested DNA samples with the control wild type and DNA markers will be separated by agarose gel electrophoresis, stained, and then analyzed.

• Nancy's son, Michael, was diagnosed at age 3 as having sarcoma. Re-cently, at age 18, he was diagnosed as having osteosarcoma.

• Nancy's other son, John (age 16), and daughter, Jessica (age 8), are free of cancer.

Valerie has fi ve children:

• Justin (age 16) • Sheila (age 14) • Robert (age 10) • Angela (age 8) • Anthony (age 6).

All the children show no signs of cancer at this time. Valerie has requested that DNA sequencing be done for each of her children (see Module Three).

Module One: Construction of a Family Pedigree


314EDVO-Kit #



The Exp

erimen

t

AGAROSE GEL REQUIREMENTS FOR THIS EXPERIMENT

• Recommended gel size: 7 x 7 cm

• Number of sample wells required: 5

• Agarose gel concentration: 0.8%

PREPARING THE AGAROSE GEL

1. Close off the open ends of a clean and dry gel bed (casting tray) by us-ing rubber dams or tape.

2. Place a well-former template (comb) in the fi rst set of notches at the end of the bed. Make sure the comb sits fi rmly and evenly across the bed.

3. To a 250 ml fl ask or beaker, add agarose powder and buffer as indicated in the Reference Tables (Appendix A) provided by your instructor. Swirl the mixture to disperse clumps of agarose powder.

4. With a marking pen, indicate the level of the solution volume on the outside of the fl ask.

5. Heat the mixture using a microwave oven or burner to dissolve the aga-rose powder.

6. Cool the agarose solution to 60°C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume marked in step 4.

After the gel is cooled to 60°C:

7. Place the bed on a level surface and pour the cooled agarose solution into the bed.

8. Allow the gel to completely solidify. It will become fi rm and cool to the touch after approximately 20 minutes.

9. After the gel is solidifi ed, be careful not to damage or tear the wells while removing the rubber dams or tape and comb(s) from the gel bed.

10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.

11. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer (refer to Table B on the instruction sheet from the Appendix provided by your instructor).


If you are unfamiliar with agarose gel preparation and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com

Important Note

Continue heating until the fi nal solution appears clear (like water) without any un-dissolved particles. Check the solution carefully. If you see "crystal" particles, the agarose is not completely dissolved.


16



EDVO-Kit #Th

e Ex

per

imen

t

LOADING THE SAMPLES

This experiment is designed for staining with InstaStain® Ethidium Bromide. The amount of sample that should be loaded is 18-20 µl. Make sure the gel is completely submerged under buffer before loading the samples and con-ducting electrophoresis.


Reminder:

Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

+Black Red

Sample wells

–

Electrophoresis can be completed in 15-20 minutes under optimal conditions. For Time and Voltage recommendations, refer to Table C (Appendix A).

LOADING DNA SAMPLES

12. Heat the DNA fragments (tubes A-E) for two minutes at 65°C. Allow samples to cool for a few minutes before gel loading.

13. Load 18-20 µl of each DNA sample in the following manner:

Lane Tube 1 A Standard DNA Fragments 2 B Control DNA 3 C Patient Peripheral Blood DNA 4 D Patient Tumor DNA 5 E Patient Breast Normal DNA

RUNNING THE GEL

14. After the DNA samples are loaded, properly orient the cover and care-fully snap it onto the electrode terminals.

15. Insert the plugs of the black and red wires into the corresponding inputs of the power source.

16. Set the power source at the required voltage and conduct electrophore-sis for the length of time determined by your instructor.

17. Check to see that current is fl owing properly - you should see bubbles forming on the two platinum electrodes.

18. After the electrophoresis is completed, disconnect the power and re-move the gel from the bed for staining.

STAINING AND VISUALIZATION OF DNA After electrophoresis, agarose gels require staining to visualize the separated DNA samples. Your instructor will provide instructions for DNA staining with InstaStain® Ethidium Bromide (Appendix C).


314EDVO-Kit #



The Exp

erimen

t

Module Three: Analysis of Autorads to Search for p53 Mutagens

In this part of the experiment, x-ray results of the wild p53 and samples from Valerie's fi ve children will be read to determine whether or not there are mutations.

Valerie's children Ages Autorad #

Justin 16 1 Sheila 14 2 Robert 10 3 Angela 8 4 Anthony 6 5

1. For each of Valerie's children, obtain the appropriate sample autoradio-graph and place it on a light box to enhance visualization.

2. The sequencing reactions have all been loaded in order: G-A-T-C.

3. Begin analysis of the DNA sequence at the bottom of the autoradio-graph with the circled band, which is an A.

4. Compare the deduced sequence to the wild type sequence shown in the box below.

5. Identify the location of the potential mutant nucleotide. What was the mutation? Is there more than one mutation?

6. Based on the information obtained from the x-rays, which of Valerie's children have a mutation in their DNA sequence?

Note: This is a simulation and the DNA sequence is not that of p53. The principles of reading DNA sequences and fi nding the point mutation is the same.

Wild Type sequence:

5'-AGCTTGGCTGCAGGTCGACGGATCCCCAGGAATTCGTAAT-3'


18



EDVO-Kit #Th

e Ex

per

imen

t

Answer the following study questions in your laboratory notebook or on a separate worksheet.

1. What is the difference between tumor suppressors and oncogenes?

2. What are the effects of hot spots in p53 protein structure?

3. Why does Valerie's tumor DNA sample have fewer bands than the pe-ripheral blood?

4. What is the purpose of the control lane?

5. Can a physician proceed with diagnosis based on molecular biology data?

Study Questions

31

314EDVO-Kit #Material Safety Data Sheets

Full size (8.5 x 11”) pdf copy of MSDS available at www.edvotek.com or by request.

Mat

eria

l Saf

ety

Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

's H

azar

d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

00 S

tan

dar

d m

ust

be

con

sult

ed f

or

spec

ific

req

uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

nd

Lis

t)N

ote

: B

lan

k sp

aces

are

no

t p

erm

itte

d.

If a

ny

item

is n

ot

app

licab

le, o

r n

o in

form

atio

n is

ava

ilab

le, t

he

spac

e m

ust

b

e m

arke

d t

o in

dic

ate

that

.

Sect

ion

IM

anu

fact

ure

r's

Nam

e

Sect

ion

II -

Haz

ard

ou

s In

gre

die

nts

/Id

enti

fy In

form

atio

n

Emer

gen

cy T

elep

ho

ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

on

al)

Ad

dre

ss (

Nu

mb

er, S

tree

t, C

ity,

Sta

te,

Zip

Co

de)

EDV

OTE

K, I

nc.

1467

6 R

oth

geb

Dri

veR

ock

ville

, MD

208

50

Haz

ard

ou

s C

om

po

nen

ts [

Spec

ific

C

hem

ical

Iden

tity

; C

om

mo

n N

ame(

s)]

O

SHA

PEL

AC

GIH

TLV

Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

g P

oin

tSe

ctio

n II

I - P

hys

ical

/Ch

emic

al C

har

acte

rist

ics

Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

s

Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

Pres

sure

(m

m H

g.)

Vap

or

Den

sity

(A

IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

Gra

vity

(H

0 =

1)

2

Ag

aro

se

10/0

5/06

This

pro

du

ct c

on

tain

s n

o h

azar

do

us

mat

eria

ls a

s d

efin

ed b

y th

e O

SHA

Haz

ard

Co

mm

un

icat

ion

Stan

dar

d.

CA

S #9

012-

36-6

For

1% s

olu

tio

n 1

94 F

N

o d

ata

N

o d

ata

No

dat

a

No

dat

a

No

dat

a

Inso

lub

le -

co

ld

W

hit

e p

ow

der

, no

od

or

N.D

. = N

o d

ata

No

dat

a

N

.D.

N.D

.

Wat

er s

pra

y, d

ry c

hem

ical

, car

bo

n d

ioxi

de,

hal

on

or

stan

dar

d f

oam

Poss

ible

fir

e h

azar

d w

hen

exp

ose

d t

o h

eat

or

flam

e

No

ne

ED

VO

TE

K®

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

G

en. d

iluti

on

ven

tila

tio

n

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

Yes

Sp

lash

pro

of

go

gg

les

Imp

ervi

ou

s cl

oth

ing

to

pre

ven

t sk

in c

on

tact

No

neX

N

on

e

No

dat

a av

aila

ble

X

No

ne

Yes

Y

es

Yes

Inh

alat

ion

: N

o d

ata

avai

lab

le

In

ges

tio

n:

Larg

e am

ou

nts

may

cau

se d

iarr

hea

No

dat

a av

aila

ble

No

dat

a av

aila

ble

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Swee

p u

p a

nd

pla

ce in

su

itab

le c

on

tain

er f

or

dis

po

sal

No

rmal

so

lid w

aste

dis

po

sal

No

ne

No

ne

Ch

emic

al c

artr

idg

e re

spir

ato

r w

ith

fu

ll fa

cep

iece

.

ED

VO

TE

K®

Mat

eria

l Saf

ety

Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

's H

azar

d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

00 S

tan

dar

d m

ust

be

con

sult

ed f

or

spec

ific

req

uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

nd

Lis

t)N

ote

: B

lan

k sp

aces

are

no

t p

erm

itte

d.

If a

ny

item

is n

ot

app

licab

le, o

r n

o in

form

atio

n is

ava

ilab

le, t

he

spac

e m

ust

b

e m

arke

d t

o in

dic

ate

that

.

Sect

ion

IM

anu

fact

ure

r's

Nam

e

Sect

ion

II -

Haz

ard

ou

s In

gre

die

nts

/Id

enti

fy In

form

atio

n

Emer

gen

cy T

elep

ho

ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

on

al)

Ad

dre

ss (

Nu

mb

er, S

tree

t, C

ity,

Sta

te,

Zip

Co

de)

EDV

OTE

K, I

nc.

1467

6 R

oth

geb

Dri

veR

ock

ville

, MD

208

50

Haz

ard

ou

s C

om

po

nen

ts [

Spec

ific

C

hem

ical

Iden

tity

; C

om

mo

n N

ame(

s)]

O

SHA

PEL

AC

GIH

TLV

Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

g P

oin

t

Sect

ion

III -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

s

Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

s

Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

Pres

sure

(m

m H

g.)

Vap

or

Den

sity

(A

IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

Gra

vity

(H

0 =

1)

2

50x

Elec

tro

ph

ore

sis

Bu

ffer

This

pro

du

ct c

on

tain

s n

o h

azar

do

us

mat

eria

ls a

s d

efin

ed b

y th

e O

SHA

Haz

ard

Co

mm

un

icat

ion

Sta

nd

ard

.

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

Ap

pre

ciab

le, (

gre

ater

th

an 1

0%)

Cle

ar, l

iqu

id, s

ligh

t vi

neg

ar o

do

r

No

dat

a

N.D

. = N

o d

ata N.D

.

N.D

.

Use

ext

ing

uis

hin

g m

edia

ap

pro

pri

ate

for

surr

ou

nd

ing

fir

e.

Wea

r p

rote

ctiv

e eq

uip

men

t an

d S

CB

A w

ith

fu

ll fa

cep

iece

op

erat

ed in

po

siti

ve p

ress

ure

mo

de.

No

ne

iden

tifi

ed

10/0

5/06

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

N

on

e

Stro

ng

oxi

diz

ing

ag

ents

Car

bo

n m

on

oxi

de,

Car

bo

n d

ioxi

de

X

N

on

e

Yes

Y

es

Y

es

No

ne

No

ne

iden

tifi

ed

Irri

tati

on

to

up

per

res

pir

ato

ry t

ract

, ski

n, e

yes

No

ne

Ing

esti

on

: If

co

nsc

iou

s, g

ive

larg

e am

ou

nts

of

wat

er

Eyes

: Fl

ush

wit

h w

ater

In

hal

atio

n:

Mo

ve t

o f

resh

air

Sk

in:

Was

h w

ith

so

ap a

nd

wat

er

Wea

r su

itab

le p

rote

ctiv

e cl

oth

ing

. M

op

up

sp

ill

and

rin

se w

ith

wat

er, o

r co

llect

in a

bso

rpti

ve m

ater

ial a

nd

dis

po

se o

f th

e ab

sorp

tive

mat

eria

l.

Dis

po

se in

acc

ord

ance

wit

h a

ll ap

plic

able

fed

eral

, sta

te, a

nd

loca

l en

viro

men

tal r

egu

lati

on

s.

Avo

id e

ye a

nd

ski

n c

on

tact

.

No

ne

Yes

N

on

e

Yes

N

on

e

Yes

_Saf

ety

go

gg

les

No

ne

No

ne

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

N

on

e

Stro

ng

oxi

diz

ing

ag

ents

Car

bo

n m

on

oxi

de,

Car

bo

n d

ioxi

de,

nit

rog

en o

xid

es, h

ydro

gen

bro

mid

e g

as

X

N

on

e

Yes

Y

es

Yes

No

dat

a av

aila

ble

Irri

tati

on

to

mu

cou

s m

emb

ran

es a

nd

up

per

res

pir

ato

ry t

ract

No

dat

a

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Wea

r SC

BA

, ru

bb

er b

oo

ts, r

ub

ber

glo

ves

Mix

mat

eria

l wit

h c

om

bu

stib

le s

olv

ent

and

bu

rn in

a c

hem

ical

inci

ner

ato

r eq

uip

ped

aft

erb

urn

er a

nd

scr

ub

ber

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Mu

tag

en

Yes

Ch

em. f

um

e h

oo

d

No

N

on

e

Ru

bb

er

C

hem

. saf

ety

go

gg

les

R

ub

ber

bo

ots

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Acu

te: M

ater

ial i

rrit

atin

g t

o m

uco

us

mem

bra

nes

, up

per

res

pir

ato

ry t

ract

, eye

s, s

kin

Ch

ron

ic:

May

alt

er g

enet

ic m

ater

ial

SCB

A

Mat

eria

l Saf

ety

Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

's H

azar

d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

00 S

tan

dar

d m

ust

be

con

sult

ed f

or

spec

ific

req

uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

nd

Lis

t)N

ote

: B

lan

k sp

aces

are

no

t p

erm

itte

d.

If a

ny

item

is n

ot

app

licab

le, o

r n

o in

form

atio

n is

ava

ilab

le, t

he

spac

e m

ust

b

e m

arke

d t

o in

dic

ate

that

.

Sect

ion

IM

anu

fact

ure

r's

Nam

e

Sect

ion

II -

Haz

ard

ou

s In

gre

die

nts

/Id

enti

fy In

form

atio

n

Emer

gen

cy T

elep

ho

ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

on

al)

Inst

aSta

in, I

nc.

P.O

. Bo

x 12

32W

est

Bet

hes

da,

MD

208

27

Haz

ard

ou

s C

om

po

nen

ts [

Spec

ific

C

hem

ical

Iden

tity

; C

om

mo

n N

ame(

s)]

O

SHA

PEL

AC

GIH

TLV

Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

g P

oin

t

Sect

ion

III -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

s

Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

s

Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

Pres

sure

(m

m H

g.)

Vap

or

Den

sity

(A

IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

Gra

vity

(H

0 =

1)

2

Inst

aSta

in®

Eth

idiu

m B

rom

ide

Eth

idiu

m B

rom

ide

D

ata

no

t av

aila

ble

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

Solu

ble

Ch

emic

al b

ou

nd

to

pap

er, n

o o

do

r

No

dat

a

N.D

. = N

o d

ata N.D

. N

.D.

Wat

er s

pra

y, c

arb

on

dio

xid

e, d

ry c

hem

ical

po

wd

er, a

lco

ho

l or

po

lym

er f

oam

Wea

r p

rote

ctiv

e cl

oth

ing

an

d S

CB

A t

o p

reve

nt

con

tact

wit

h s

kin

& e

yes

Emit

s to

xic

fum

es

10/0

5/06

CA

S# 1

39-3

3-3

(2,7

-Dia

min

o-1

0-Et

hyl

-9-P

hen

ylp

hen

anth

rid

iniu

m B

rom

ide)

ED

VO

TE

K®

the biotechnology education company p d a t e d r e v i s e d a d n the biotechnology education...

Documents