Recombinant DNA and

Recombinant DNA and
Genetic Engineering

Chapter 12

Goals for this chapter


Have a general understanding of
Genetic Engineering and Biotechnology



Be able to form an opinion that is based
on fact or emotion


And recognize the difference

Focus our attention on


Genetic Improvement of Crop Plants



Other possible topics


Genetic improvement of animals


Genetic improvement of Humans


Stem cell therapy


Forensics


Historical research


And on and on


What is Biotechnology


Definitions



Biotechnology


Genetic Engineering


Recombinant DNA


Transgenic or GMO

Biotechnology


Genetic Improvement of plants and
animals


This would include the activities of Plant
Breeders



And plant breeders are really nice people


Genetic Changes


Humans have been changing the genetics
of other species for thousands of years


Artificial selection of plants and animals


Natural processes also at work


Mutation, crossing over

Traditional plant breeding


Select parents


Cross and generate variable offspring


Select the desired types


Test
-

Test
-

Test


Multiply


Release


7 to 15 years


Norman Borlaug


Nobel Peace Prize


1970


The Green Revolution




http://nobelprize.org/peace/laureates/

1970/borlaug
-
lecture.html

Barbara Mclintock


Transposons


1983 Nobel Prize

in Medicine

Genetic Engineering


Modern Biotechnology
-

which uses the
knowledge of DNA to manipulate the genetic
makeup of an organism



Recombinant DNA
-

take a gene from one
organism and place it into another organism



Transgenic or GMO
-

an organism that
contains DNA from another organism


Would You Eat a

Genetically Engineered

Food?

Most GMO
’
s are Plants


34% of Corn


71% of Cotton


75% of soybeans



Silk is Soy

Why no GMO

Until recently the terms Genetically Modified
Organism (GMO), GMO
-
Free and Non
-
GMO
were used to help identify foods that
contained genetically altered ingredients.


These terms are no longer recognized by the
Food and Drug Administration (FDA) and
therefore cannot be used on food packaging.


What about Tostito
’
s


Or Pepsi


Or Coke


Or CapN Crunch???

How to Manipulate DNA

in the Lab

Examples of Transformation


Natural Systems


Bacteria


Viruses


Bacterium

bacterial

chromosome

plasmid

Transformation with DNA fragment

bacterial

chromosome

DNA

fragments


Virus enters host cell.

2

virus

viral DNA

host cell

host cell DNA

“hybrid virus”

viral proteins

viral DNA


Virus attaches to

susceptible host cell.

1


Virus releases its DNA into

host cell; some viral DNA (red)

may be incorporated into the

host cell’s DNA (blue).

3


Viral genes encode synthesis

Of viral proteins and viral gene

Replication. Some host cell DNA

May attach to replicated viral

DNA (red/blue).

4


New viruses assemble; host

cell DNA is carried by “hybrid

viruses.”

5


Host cell bursts, releasing

newly assembled viruses.

when “hybrid viruses” infect a

second cell, they may transfer

genes from the first cell to the

second cell.

6

GE Tool Box


Restiction Enzymes


Cloning Vectors


cDNA Cloning


Reverse
Transcriptase


PCR


Gene Library
(Isolation)


Transformation of
Plants


Based on the Central Dogma and the fact that in
virtually organisms the CODE is perfectly conserved
almost

Amplifying DNA


Fragments can be inserted into

fast
-
growing microorganisms



Polymerase chain reaction (PCR)

Polymerase Chain Reaction


Sequence to be copied is heated


Primers are added and bind to ends of
single strands


DNA polymerase uses free nucleotides
to create complementary strands


Doubles number of copies of DNA

Polymerase
Chain
Reaction

Double
-
stranded

DNA to copy

DNA heated to

90
°
–

94
°
C

Primers added to

base
-
pair with

ends

Mixture cooled;

base
-
pairing of

primers and ends

of DNA strands

DNA polymerases

assemble new

DNA strands

Fig. 16
-
6, p. 256

Stepped Art

Polymerase
Chain
Reaction

Stepped Art

Mixture heated again;
makes all DNA
fragments unwind

Mixture cooled; base
-
pairing between
primers and ends of
single DNA strands

DNA polymerase
action again
doubles number of
identical DNA
fragments


Fig. 16
-
6, p. 256

Fig. 16
-
7, p.247

DNA Fingerprinting

Guilty or Innocent

8 side
-
by
-
side (tandem) repeats

of the same 4
-
nucleotide sequence,


Nylon paper with DNA is bathed in a solution of labeled DNA probes (red) that


are complementary to specific DNA segments in the original DNA sample.


Complementary DNA segments are labeled by probes

(red bands).

nylon paper

solution of DNA probes (red)

gel

gel

power supply

DNA bands

(not yet visible)

�

�

wells

pipetter

nylon

paper

DNA samples are pipetted into wells

(shallow slots) in the gel. Electrical current

is sent through the gel (negative at end

with wells, positive at opposite end).

Electrical current moves DNA

segments through the gel. Smaller

pieces of DNA move farther toward the

positive electrode.

Gel is placed on special nylon

paper. Electrical current drives

DNA out of gel onto nylon.

�

�

gel

power supply

�

�

wells

pipetter

DNA samples are pipetted into wells

(shallow slots) in the gel. Electrical current

is sent through the gel (negative at end

with wells, positive at opposite end).

�

�

DNA bands

(not yet visible)

�

�

Electrical current moves DNA

segments through the gel. Smaller

pieces of DNA move farther toward the

positive electrode.

�

�

gel

�

�

nylon

paper

Gel is placed on special nylon

paper. Electrical current drives

DNA out of gel onto nylon.

�

�


Nylon paper with DNA is bathed in a solution of labeled DNA probes (red) that


are complementary to specific DNA segments in the original DNA sample.

nylon paper

solution of DNA probes (red)

�

�

�

�


Complementary DNA segments are labeled by probes

(red bands).

�

�

�

�

STR name

Penta D

CSF

D16

D7

D13

D5

D16: an STR on chromosome 16

DNA samples from

13 different people

Number of repeats

STR name

Penta D

CSF

D16

D7

D13

D5

Number of repeats

D16: an STR on chromosome 16

DNA samples from

13 different people

Number of repeats

Genetic Engineering

Transformation of Plants


Genes are isolated, modified, and
inserted into an organism


Made possible by recombinant
technology


Cut DNA up and recombine pieces


Amplify modified pieces

Process

Board Diagram


Corn Transformation with RR gene

From a bacteria or petunia

Hypothetical Situation


Corn Cotton and Alfalfa

Roughly 400 million people in the world today are at risk of Vitamin A
deficiency, which already affects 100
-
200 million children. Vitamin A
deficiency causes various health problems, including blindness. Because
rice is an important crop, eaten by almost half of the people in the world,
the Rockefeller Foundation and the European Union funded research
into varieties that might offer global health benefits.


It may now be possible, thanks to agricultural biotechnology, to make rice
and other crops into additional sources of Pro
-
Vitamin A. With
Monsanto's help, the developers of "Golden Rice" and mustard with more
Pro
-
Vitamin A should one day be able to deliver their gift of better
nutrition to the developing nations of the world through staple crops
readily available to poor and vulnerable populations


Imagine sharing science to help others develop crops that could help
reduce Vitamin A deficiency, a leading cause of blindness and infection
among the young.



Imagine innovative agriculture that creates incredible things.

Discussion


Debate the Merits or Dangers of Golden
Rice.



Group One
-

Make your case in front of
Congress to obtain money to distribute
this rice (as rice seed) to the farmers of
Southeast Asia and Africa


Group Two
-

Argue against this.

You be the judge


With your pocket book


Or your advocacy


Ethical Issues


Who decides what should be “corrected”
through genetic engineering?


Should animals be modified to provide
organs for human transplants?


Should humans be cloned?