Day 4: Genetics

tanktherapistBiotechnology

Oct 23, 2013 (3 years and 7 months ago)

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Day 4: Genetics

Selcen Guzey and Tamara Moore

Agenda


Genetically modified crops

-
DNA extraction

-
PCR

Activity 1: PCR with paper clips


Online resources
-
NIH

Activity 2: Genetically modified sugar beet
-
Debate

-
Gel electrophoresis

GMO Investigator Kit from
BioRad


Part I: DNA extraction (30 minutes)


Part II: PCR (~4 hours)


Part III: Gel electrophoresis (30 minutes)


Part IV: Analyzing results (30 minutes)

This kid provides an investigation in which you will extract DNA from food samples,

amplify genetics sequences using PCR, and use gel electrophoresis to identify the

presence or absence of the amplified marker sequences.

Pre
-
lab Discussion



What is a GM crop?


A plant that has been genetically modified by the insertion of foreign
genetic material (pest resistance, herbicide tolerance, delayed fruit
ripening, improved fruit yield, increased nutrient content etc.)



How does one test foods and crops to identify which contain GM
genomes?

ELISA

(
E
nzyme
l
inked
i
mmuno
s
orbent

a
ssay) identifies specific proteins
produced by GM plants.


PCR

(
P
olymerase
c
hain
r
eaction)


Identify DNA sequences resulting from genetic modification


(35 Promoter and Terminator from
Agrobacterium

tumefaciens
)


Part I: DNA Extraction


Each group will extract DNA from a control non
-
GMO food and a grocery store food item.

Control non
-
GMO food
: Certified non
-
GMO grain from
BioRad

Grocery store food samples
: Cheese puffs, soy
-
based bacon
bits, corn nuts, granola bars, corn cereal, tortilla chips, fresh
corn, corn meal, organic cereal, potato chips


Weight your food sample, grind it with water,
add a tiny amount of it to a
srewcap

tube
containing
InstaGene

matrix and boil it for 5
minutes.


Part I: DNA Extraction

Material

Quantity

Screwcap

tube

2

Beaker of distilled water

1

Food

sample

2

Disposable

plastic transfer
pipets

2

2
-
20

µ
l

micropipet

(for non
-
GMO

food
cont)

1

2
-
20µl
pipet

tips

1 rack

Mortar

and pestle

1

Part II: PCR


How might PCR be used to determine if a food
sample has been genetically modified?

PCR can be used to detect foreign DNA in
genetically modified food.

PCR allows us to amplify specific sections of
DNA and make millions of copies of the
target sequence.


Part II: PCR


This part of the experiment is to determine whether or
not the DNA you extracted from food samples contains or
does not contain the target sequences of interest typically
found in GM foods.


In the first part, you extracted DNA from a certified non
-
GMO food and a test food sample. In this part, you will
prepare those two samples and a positive control (GMO
positive templates DNA) for the PCR.



Material

Quantity

Ice bath containing 3
tubes

1

GMO

master mix (red)

1

Plant

master mix (green)

1

GMO

positive cont. DNA

1

Test

food DNA

1

PCR

tubes

6

Foam
microtube

holder

1


2
-
20µl
micropipet


2
-
20µl
pipet

tips

1

1 rack

PCR Tubes

Tube

#

DNA

Master Mix

1

20µl Non
-
GMO

food control DNA

20µl Plant master mix (green)

2

20µl Non
-
GMO

food control DNA

20µl GMO master

mix (red)

3

20µl Test

food DNA

20µl Plant master mix (green)

4

20µl Test

food DNA

20µl GMO master

mix (red)

5

20µl GMO

positive control

20µl Plant master mix (green)

6

20µl GMO

positive control

20µl GMO master

mix (red)

1) You will set up 2 PCR reactions for each DNA sample, which makes 6 PCR reactions in total.

One PCR, using the plant primers, is a control to whether plant DNA was successfully extracted

from the samples. Plant primers amplify a section of a chloroplast gene (455
bp
)


2) The second PCR reaction is to determine whether or not the food samples contains GM
sequences. GMO primers identify DNA sequences that are common to (85%) GM plants (203
bp

and 225
bp
)


3)The non
-
GMO food is the indicator of false positive and the GMO positive template control
is an indicator of false negative.


PCR

PCR is developed by
Kary

Mullis in 1983.


It relies on thermal cycling, consisting of cycles of repeated heating
and cooling of the reaction for DNA replication. DNA template is
exponentially

amplified.


PCR requires several components:


DNA template


Primers that contains sequences complementary to the target
region


DNA polymerase (
Taq

DNA polymerase from
Thermus

aquaticus
)


Nucleotides


Buffer solution


How is PCR used?


PCR impacted several areas of genetic
research:



PCR used as a medical diagnostic tool to detect
specific mutations that may cause genetic disease



PCR used in criminal investigations and courts of
law to identify suspects



PCR used in the sequencing of the human genome


PCR

Denaturation
: DNA is heated to separate
strands (94

°
C for 1 minute)

Annealing
: The mixture is cooled and primers
bind to strands (60

°
C for 1 minute)

Elongation
: DNA polymerase adds nucleotides
to strands, producing 2 complementary
strands (72
°
C for 2 minutes)


The procedure is repeated again!

PCR Simulation

http://learn.genetics.utah.edu/content/labs/pcr
/


Generation of precise
-
length
fragments

Original long template DNA molecules are never
fully duplicated

After each cycle, two intermediate
-
length
strands are produced (they cannot
exponentially amplified)

The precise
-
length strands generated from the
intermediate strands are amplified
exponentially.

PCR with Paper Clips


In this activity you will be performing PCR
using paper clips. Each colored paper clip
represents a particular nucleotide in the DNA:

Blue
:
Adenin

Red
: Thymine

Green
: Guanine

Yellow
: Cytosine

Online Resources

NHI


http://ghr.nlm.nih.gov/


http://www.ncbi.nlm.nih.gov/education/


Human molecular genetics book:
http://www.ncbi.nlm.nih.gov/books/NBK7580
/


BLAST: Basic Local Alignment Search Tool

http://blast.ncbi.nlm.nih.gov/Blast.cgi


Breast Cancer Susceptibility 1 and 2
Genes

For every 1000 women….


120 (12%) will get breast cancer


6 of them inherited mutations in BRCA1 or BRCA 2. BRCA 1 and
BRCA 2 mutations account for about 5% of all breast cancer.
This may seem like a small number, but a woman who
inherits a cancer causing BRCA1 or BRCA2 mutation, her risk
of developing breast cancer is up to 85%.


The remaining cases are caused by mutations in other genes,
environmental exposures, and other unknown factors.

Breast Cancer Tests

How can we diagnose BRCA 1 and BRCA 2
mutations?


The doctor would isolate DNA from the patient’s
blood and sequence the BRCA 1 and 2 genes
to determine whether known cancer
-

causing
mutations are present.

National Center for Biotechnology
Information (NCBI)

1) Go to
http://www.ncbi.nlm.nih.gov/

2) Type BRCA1 into the Search box

3) Click the “All resources”

4) Click the “Map viewer”

5) Select home sapiens and click go

6) Choose chromosome 17

7) Click BRCA1

8) Reference sequences under the table of contents

9) Click the FASTA


BLAST

Can be used to:


-
Compare 2 or more DNA
sequences to each other

-
Compare 2 or more protein
sequences to each other

-
Compare a single sequence to
many sequences found in
databases


BLAST


Go to the NCBI blast website
http://blast.ncbi.nlm.nih.gov/Blast.cgi


Select nucleotide blast


Click to “Align two or more sequences”


Copy reference sequence for BRCA1


Copy the DNA sequence from the patient


Click BLAST


Click formatting options, find the Alignment view and
choose “Query
-
anchored dots with identities”


Click reformat


GM Crops


Bt
corn
,

Bt
cotton, and Bt potato

(resistant to the Bacillus
thuringiens
is

(Bt) toxin)



Roundup
Ready
soybeans and


sugar beet

(tolerant to the herbicide
-
glyphosate
)


Golden
rice


(synthesize beta
-
carotene)


Flavr

Savr

tomato



(slow the ripening process)


UH
Rainbow
papaya

(resistant to plant virus disease)


How do you genetically modify a crop?

1)
Identify a protein that has the potential to
improve a crop.

Bt crops has a gene from Bacillus
thuringiensis

(Bt).
They produce delta
-
endotoxin

that is lethal to
corn borers, a common pest on corn plants.

2) Isolate the gene that codes for the protein

3) Engineer the gene so that the crop plant’s cells
can read it correctly
-

removing
introns
, adding
promoter (CaMV35) and terminator from
Agrobacterium

tumefaciens
.


How do you genetically modify a crop?

Inducing the gene into the plant:



1) Cells are removed from the parent plant, they grow on a special
medium
-
Single cells, plant cells without cell walls and pieces of leaves
can often be used to generate a new plant on culture media given the
required nutrients and hormones.

2) The gene then transferred into the plant cells by:


-

electric shock/
electroporation


-

heat shock


-
gene gun


-
Agrebacterium

tumefaciens


This bacterium causes crown gall disease by


inserting some of its DNA to a plant cell.



3) The transformed cells are isolated and then induced with plant hormones
to differentiate and grown into complete plants.


The process is inefficient, costly, and time consuming!

Sugar Beet Debate


Read the article individually and then share what you
have learned with your group members.

As a group you will be asked to address the advantages
and disadvantages of three options
from the viewpoint
of your assigned stakeholder
.

1. Issue a partial deregulation for the interim to allow
planting in certain geographic areas and/or under
certain conditions

2. Wholly deregulate the HT sugar beet, allowing it to be
grown anywhere anytime

3. Refuse to deregulate HT sugar beet, allow for farmers
to harvest existing beets, but no more planting.

Sugar Beet Debate

1) Introduce yourself as your character

2) Which option would your assigned stakeholder
advocate? If you pick (1) the partial deregulation
option, describe the terms of the partial
deregulation (geographic and conditions).

3) Support your option in terms of scientific,
economic, and cultural justifications.

4) What are the potential adverse effects of the
proposed options?


Benefits and Risks

Genetic modification of crops can produce four general
benefits:


1) agricultural
-
increased yield
,

2
) environmental
-
reduced use
of pesticides
, herbicides
,
and
fuel,

3) nutritional
-
improved quality
of foods
,
and

4) disease prevention
-

foods that work like edible
vaccines
.


Such
risks may include:



Exposure
to possible allergens and toxins, harm to the
environment
, antibiotic resistance, and
the
spread of
introduced genes
to non
-
target plants
by out
-

crossing and
pollen drift

Other Debates








GMO corn and Monarchs

Possible reading assignments:

1) Anthony M. Shelton and Mark K. Sears. (2001) The monarch
butterfly controversy: scientific interpretations of a
phenomenon. The Plant Journal 27(6), 483
-
488.

2)
Losey
, J.,
Raynor
, L. and Carter, M.E. (1999) Transgenic
pollen harms Monarch larvae. Nature, 399, 214.


Regulation of Golden Rice

Possible reading assignment:

Potrykus
, I. 2010. Regulation must be revolutionized. Nature
466: 561.

How can we check whether the PCR
generated the anticipated DNA
fragment?


Gel Electrophoresis

Gel Electrophoresis


Gel electrophoresis separates DNA molecules
based on charge and size.


DNA is negatively charged and is repelled by
the negative electrode (cathode) and
attracted by the positive electrode (anode)
when an electric current applied across the
gel.


Longer DNA fragments move more slowly than
shorter fragments.

Gel Electrophoresis


What results do you expect in each line?


Lane

Sample

Expect
Band?

Sample 1

Non
-

GMO food control with
plant primers

Sample

2

Non
-
GMO

food control with
GMO primers

Sample 3

Test

food with plant primers

Sample 4

Test

food with GMO primers

Sample 5

GMO positive control DNA
with

plant primers

Sample 6

GMO

positive control DNA
with GMO primers

Plant primers: 455
bp

GMO primers: 200bp

Analyzing Results


Step1: Check Plant PCR (Lane 5)


Step 2: Check GMO PCR (Lane 6)*


Step 3: Check DNA was extracted from non
-
GMO
food (Lane 1)


Step 4: Check PCR reactions are not
contaminated (Lane 2)*


Step 5: Check DNA was extracted from test food
(Lane 3)


Step 6: Is test food GMO positive (Lane 4)?


Analyzing Results

Analyzing Results

Analyzing Results



What was your test food?


Is your test food GMO positive?