Biotechnology

whipmellificiumBiotechnology

Feb 20, 2013 (4 years and 1 month ago)

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Chapter 13

Biotechnology

How was Ruffin’s innocence
proved?

GUILTY or INNOCENT?

Traditional Applications of Biotechnology


date back to over 10,000 years ago


Use of yeast to produce beer and wine in Egypt and Near
East


Selective breeding of plants


Selective breeding of animals

Biotechnology

is

applied biology
(broadest sense)

Any use or alteration of organisms, cells, or biological
molecules to achieve specific practical goals

13.1 What Is Biotechnology?


A
modern

emphasis on
genetic engineering
:
refers to
the modification of genetic material to achieve specific goals




major goals including


--

learning more about cellular processes:


inheritance and gene expression



--

providing better understanding and treatment of disease



--

generating economic and social benefits




A key tool:

recombinant DNA technology




transgenic

or
genetically modified organisms (GMOs)


associated techniques for
analyzing biological molecules
,
especially DNA and protein



Selective breeding

is an important tool biotechnology


13.2.1
Sexual Reproduction
recombines DNA


Due to crossing over during meiosis, each
chromosome in a gamete contains a mixture of alleles
from the two parental chromosomes


Thus, eggs and sperm contain recombinant DNA

13.2 How Does DNA Recombine in
Nature?

13.2.2

Transformation

May Combine DNA

from Different Bacterial Species

plasmid

bacterial

chromosome

Bacterium

1 micrometer

Plasmid: 1
-
100 kbp

(
E. coli

around 4,600 kbp)

DNA

Fragments

bacterial

chromosome

Transformation with
DNA fragment

bacterial

chromosome

Transformation with
plasmid

plasmid

DNA fragment is
incorporated into
chromosome

Plasmid replicates
in cytoplasm

Size range:
1000

100,000
nucleotides long

A single bacterium may contain dozens or
even hundreds of copies of a plasmid

(carry 1
-
10 kbp
DNA fragment)

What use are plasmids?

Genes carried by plasmids allowing


the bacteria carry them to
grow in novel environments


the bacteria
metabolize unusual energy sources


cause disease symptoms
, such as diarrhea, in the animal
or other organism that the bacterium infects


enable bacteria to
grow in the presence of antibiotics


13.2.3

Viruses May Transfer DNA
Between
Species


Viral life cycle

1.
Viral particle invades host cell

2.
Viral DNA is replicated

3.
Viral protein molecules are synthesized

4.
Offspring viruses are assembled and break out of the
host cell



Viral transfer of DNA

Viruses may package some genes from host cell into viral
particles during assembly




Infection of new host cell injects genes from previous host,
allowing for recombination



Virus releases its DNA into
host cell; some viral DNA (red)
may be
incorporated

into the
host cell’s DNA (blue).



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
.


hybrid virus”



New viruses assemble; host

cell DNA is carried by “
hybrid

viruses
.”



Virus enters host cell.

virus

viral DNA

host cell

host cell DNA



Virus attaches to

susceptible host cell.



Viral genes encode synthesis
of viral proteins and viral gene

replication. Some host cell DNA

may attach to replicated viral

DNA (red/blue).

viral proteins

viral DNA

Biotechnology and Forensics


Forensics

is the science of criminal and victim identification


DNA technology has allowed forensic science to identify victims
and criminals from trace biological samples


Genetic sequences of any human individual are unique


DNA analysis reveals patterns that identify people with a high
degree of accuracy

13.3 How Is Biotechnology Used in
Forensic Science?

13.3.1 The
P
olymerase
C
hain
R
eaction
Amplifies DNA

Developed by Kary B. Mullis of the Cetus Corporation in 1986

Acquired the Nobel Prize for Chemistry in 1993.


Forensic technicians typically have very little DNA with which to
perform analyses


Polymerase Chain Reaction

(
PCR
) produces virtually unlimited
copies of a very small DNA sample


PCR requires small pieces of DNA (called
primers
) that are
complementary to the
gene sequences

targeted

for copying


A PCR “run” is basically DNA replication in a tiny test tube


Template DNA
,
primer
,
nucleotides
, and
DNA polymerase

are all in the reaction mix

PCR copies a specific DNA sequence

One PCR cycle

original

DNA

50
°
C



primer

to
form complementary
base pairs with DNA

72
°
C



make copies
of the DNA segment


DNA

polymerase

new DNA

strands

primers

1


Heating

separates

DNA strands.

2


Cooling allows

primers and DNA
polymerase to bind.

3


New DNA

strands are

synthesized.

90
°
C



break
the H
-
bonds

4

Repetition of the cycle

1

2

3

1

2

4

8

PCR cycles



(b) Each PCR cycle doubles the number of copies of the DNA

4

16

etc.


etc.

DNA fragment

to be amplified

DNA copies

20 cycles


about 1 million copies

30 cycles


about 1 billion copies

The
Choice of Primers

Determines Which
Segments of DNA Are Amplified

How would a forensics lab know which primers to use?



Small, repeating segments of DNA called
short tandem repeats

(
STRs
) can be used to identify people with astonishing accuracy.



2
-
5 nucleotides long and usually in introns.

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

T

A

A

T

A

T

T

A

T

A

T

A

T

A

G

C

A

T

A

T

G

C

A

T

T

A

8 side
-
by
-
side (tandem) repeats

of the same 4
-
nucleotide sequence,

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

A

T

T

A

A

T

G

C

G

C

T

A

A

T

This STR, called D5, is not part of any known gene. The sequence AGAT
may be repeated from7 to 13 times in different individuals.

Why is the copy number of an individual STR so much
variability?



STRs probably have no biological function.



As a result, DNA replication errors that lead to different numbers
of STR repeats are not selected against during evolution, and
variability has accumulated over evolutionary time.

In 1999, British and American law enforcement agencies agreed to
use a set of 10 to13 STRs each 4 nucleotides long, that vary greatly
among individuals.

Forensics use
PCR primers

that amplify only the DNA immediately
surrounding the STRs.

How does the lab determine how many repeats occurred in
their DNA samples?

13.3.2
Gel Electrophoresis

Separates and
Identifies DNA Segments

Most of sophisticated & expensive machines are based on two methods:

1.

Separating the DNA segments with different sizes by

gel
electrophoresis

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.)

2. Labeling specific DNA fragments of interest

DNA “bands”

(
not yet visible
)

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

nylon

paper

The phosphate groups in the backbones of DNA



the negatively charged DNA fragments

(
-
) electrode to (+) electrode

13.3.3
DNA Probes

Are Used to Label
Specific Nucleotide Sequences

STR #1: probe base
-
pairs and binds

DNA probe

label

(colored

molecule)

STR #2: probe cannot base
-
pair; does not bind

identify the location of
a gene sequence by
hydrogen
-
bonding to
the band containing it

How can a technician
identify a
specific

STR
?

How does nature
identify
sequences

of DNA?

nylon paper

solution of DNA

probes
(red)

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).

In modern forensic
analysis, the STRs are
usually directly labeled
with colored molecules
during the PCR and
labeled DNA probes
are not required
, the
technique called
real
-
time PCR
.

In modern STR analysis, the suspect and crime scene DNA
samples can be run on different gels, in different states or
countries.
Why?

The numbers and positions of the bands on the gel can be
determined by the numbers of repeats of each STR.



The numbers of bands for a sample:

Appeared 2 bands for one person



The STR gene is
heterozygous


How’s about a homozygous STR gene?



The position of a band shows the copy number of the DNA
fragment.

13.3.4 Every Person Has a Unique
DNA Profile

DNA samples run on STR gels produce a pattern, called
a DNA profile

STR name

Penta D

CSF

D16

D16: an STR on chromosome 16

DNA samples from

13 different people

D7

D13

D5

15

14

13

12

11

10

9

8

Number of repeats

For the first person
:

PentaD, D7 and D13: 2 bands




heterozygous genes

CSF, D16 and D5: one band




homozygous genes

For the D16 STR
:

The first person’s DNA:


12 repeats and homozygous

The second person’s DNA:


12 & 13 repeats and heterozygous

Copyright © 2005 Pearson Prentice Hall, Inc.

13.4 How Is Biotechnology Used in
Agriculture?

13.4.1 Many Crops Are Genetically Modified

Commonly modified to improve the crops’ resistance to insects & herbicides

Bt

gene


Crop plants are commonly modified to improve insect
and herbicide resistance


Herbicide resistant crops withstand applications of
weed
-
killing chemicals


Bt

gene

(from
Bacillus thuringiensis

bacterium)
can be inserted into plants to produce insect
-
killing
protein in crops

The Desired Gene Is
Cloned

Cloning a gene usually involved two tasks: (1) obtaining the gene &
(2) inserting it into a plasmid



huge numbers of gene copy

Obtaining a gene via two ways:

(1) Isolating the gene from the organism that makes it

(2) Synthesizing the gene using PCR or DNA synthesizers

Producing huge numbers of copies of the gene by inserting the
target gene into a plasmid and plasmid manipulation in bacterial
cells.

Why insert the desired gene into a plasmid?


producing huge numbers of copies of the gene simply


the gene separated from the bacteria fairly easily


plasmids may then be taken up by other bacteria or …

Restriction Enzymes Cut DNA at Specific
Nucleotide Sequences

single
-
stranded

“sticky ends”

A specific
restriction enzyme

(EcoRI) binds to
the GAATTC sequence and cuts the DNA,
creating DNA fragments with “sticky ends.”

Cutting Two Pieces of DNA with the Same Restriction
Enzyme Allows the Pieces to Be Joined Together


Cut both with the same restriction enzyme.

DNA including
Bt

gene

Ti Plasmid

Mix
Bt

gene and plasmid; add
DNA ligase to seal DNA.

Transform
Agrobacterium tumifaciens

with recombinant
plasmid
投影片

30

A. tumifaciens

plasmids

bacterial

chromosome

By manipulating the plasmids and bacteria appropriately,
biotechnologists can isolate and grow only the bacteria with the
desired plasmid.

Ti
-
質體(
Ti plasmid



T i p l a s m i d

t u m o r
-
i n d u c i n g p l a s m i d
的 縮 寫 , 這 是 一 種
土 壤 瘤 痂 桿 菌 中 特 有 的 細 菌 性 質 體 , 其 上 有 特 殊 的 致 病 基 因 ,
可 藉 由 細 菌 感 染 而 進 入 植 物 細 胞 , 造 成 植 物 的 腫 瘤 生 成 。
投 影 片

29

Plasmids Are Used to Insert the Bt Gene Into a Plant

Infect plant cell with transgenic bacterium.

plant cell

plant

chromosomes

A. tumifaciens

The
A. tumifaciens

contains the
Ti plasmid

which can infect many
plant species.

Insert
Bt

gene into plant chromosome.

Bt

gene



Every time that plant cell divides, it replicates the Ti plasmid




all of its daughter cells inherit the Ti DNA and
carry the Bt gene



Appropriate hormonal treatments

stimulate the transgenic plant
cells to divide and differentiate into entire plants.

13.4.2 Genetically Modified Plants May Be Used
to Produce Medicines

A plant could be engineered to produce harmless proteins that
are normally found in disease
-
causing bacteria or viruses.




as
vaccine

against diseases, such that p
otatoes have been
engineered to produce harmless hepatitis B virus and
E. coli

proteins, stimulating an immune response when eaten




Plants could be engineered to produce
human antibodies
,
conferring passive immunity to microbial infection merely by
eating the plant


13.4.3 Genetically Modified Animals May Be Useful
in Agriculture and Medicine



Transgenic (
g
enetically
m
odified or
GM
) animals can be engineered
by incorporating genes into chromosomes of a fertilized egg


Healthy transgenic animals are difficult to engineer


Growth hormone genes have been inserted into
pigs and fish species but some abnormalities
have been observed


Animals like sheep might be engineered to produce
medically important proteins in their milk



Resistance against pathogens



Cool resistance…..


13.5 Biotechnology and the Human
Genome



Findings


Human genome contains ~25,000 genes


New genes, including many disease
-
associated
genes have been discovered


Has determined the nucleotide sequence of all
the DNA in our entire set of genes, called the
human genome



The genes comprise 2% of all the DNA



Applications


Many genes were discovered whose functions
are completely unknown.


Allows biologists to predict the AA sequences of
the proteins…comparing those well known
proteins…


Improved diagnosis, treatment and cures of
genetic disorders or predispositions


Comparison of our genome to those of other
species will clarify the genetic differences that
help to make us human


Help us to appreciate our place in the evolution
of life on Earth

13.6 How Is Biotechnology Used for
Medical Diagnosis and Treatment?

13.6.1 DNA Technology Can Be Used to
Diagnose Inherited Disorders


Defective alleles differ from normal,
functional alleles because of differences in
nucleotide sequence.


A particular restriction enzyme may cut two different
alleles of a gene differently


--

Differences in nucleotide sequence within genes
produces different numbers of cutting sites and
different lengths of fragments


Differences in restriction enzyme fragments between
genes are known as
restriction fragment length
polymorphisms

(RFLPs)


RFLP differences are revealed in gel electrophoresis

Restriction Enzymes
May Cut Different Alleles of a
Gene at Different Locations

Why is this used?
P.262



If different people have different RFLPs, this can be
used to identify DNA samples



With diligent research and a little luck, m
edically
important alleles can sometimes be identified by
differences in the lengths of the restriction fragments
produced by cutting with a specific RE.

normal

globin allele

Mst II cuts a normal globin allele in 2 places, but cuts the

sickle
-
cell allele in 1 place.

sickle
-
cell

globin allele

Mst II

Mst II

Mst II

DNA probe

Mst II

Mst II

DNA probe

Diagnosing sickle
-
cell anemia with REs

Gel electrophoresis of globin alleles

large

small

AA

AS

SS

AA = homozygous normal

AS = heterozygote

SS = homozygous sickle
-
cell


Defective alleles can also be identified using DNA probes


DNA probing is especially useful where there are many different
alleles at a single gene locus


--

Cystic fibrosis is a disease caused by any of 32 alleles out of
1000 total possible alleles

Different Alleles May Bind to Different
DNA Probes


Arrays of single
-
stranded DNA complementary to each of the
defective alleles can be bound to filter paper

rows of
complementary

DNA segments
for various
mutant alleles

complementary DNA for normal allele



A person’s DNA is cut into small pieces, separated into single
strands & labeled.

The array is then bathed in the resulting
solution of labeled DNA fragments (blue).



Strands of DNA binding to complementary sequence on the paper
indicate presence of a defective allele in person’s genome

A cystic fibrosis diagnostic array


An expanded version of this
type of DNA analysis is
known as a
microarray



A microarray contains up to
thousands of probes for a
variety of disease
-
related
alleles


Microarray analysis has the
potential to comprehensively
identify disease susceptibility.

13.6.2 DNA Technology Can Be Used to Treat
Disease

All products are proteins which do not cure inherited disorders

13.6.2.1 Using Biotechnology to Treat Cystic Fibrosis

13.6.2.2 Using Biotechnology to Cure Severe
Combined Immune Deficiency

Homework

Homework

13.7 What Are the Major Ethical Issues of
Modern Biotechnology?

13.6.1.1 Are Foods from GMOs Dangerous to Eat?


13.6.1.2 Are GMOs Hazardous to the Environment?

13.7.1 Should Genetically Modified Organisms Be
Permitted in Agriculture?

GM Organisms in Agriculture


The goal of breeding or genetically modifying plants or
livestock is to make them more productive, efficient, or useful


Genetic modification differs from selective breeding
(“traditional biotechnology”)


--

Genetic engineering is much more rapid


--

Genetic engineering can transfer genes between species


--

Genetic engineering can produce new genes never seen
before on Earth


Benefits of genetically modified plants


--

Transgenic crops decrease applications of pesticides,
saving fuel, labor, and money

--

GM plants can be sold at a lower price due to farm savings

--

Genetically engineered crops can deliver greater amounts of
vitamins (e.g. “golden rice” which produces vitamin A)

Scientific Objections to GMOs


Safety issues from eating GMOs

Could ingestion of Bt protein in insect
-
resistant plants be
dangerous to humans?

Are transgenic fish producing extra growth hormone
dangerous to eat?

Could GM crops cause allergic reactions?


USDA now monitors GM foods for allergic potential

Toxicology study of GM plants (2003) concluded that
ingestion of current transgenic crops pose no significant
health dangers


Environmental hazards posed by GMOs


Pollen from modified plants can carry GM genes to the wild
plant population


Could herbicide resistance genes be transferred to
weed species, creating
superweeds
?


Could GM fish reduce biodiversity in the wild population if
they escape?


Reduced diversity in wild fish makes them more
susceptible to catastrophic disease outbreaks


US found to lack adequate system to monitor changes in
ecosystem wrought by GMOs (National Academy of
Science Study 2003)


Should parents be given information about the genetic health
of an unborn fetus?


Should parents be allowed to select the genomes of their
offspring?


Embryos from
in vitro

fertilization are currently tested
before implantation


Many unused embryos are discarded


Should parents be allowed to design or correct the genomes
of their offspring?

13.7.2 Should the Genome of Humans Be Changed
by Biotechnology?

screening

Today
,

Bone marrow failure…….

Allowing physicians only to select among existing embryos,
not to change their genomes.

Ending

amniocentesis

vagina

placenta

uterus

fetus

amniotic

fluid

chorionic villi

chorionic

villus sampling


(by suction)

cells:

sex

determination,

biochemical and

enzymatic studies

cell culture: biochemical

studies, chromosomal

analysis, analysis using

recombinant DNA

methods

centrifuge

fluid:

composition

analysis

Prenatal Genetic Screening