Pre AP Biology Chapter 16 – Recombinant DNA and Genetic ...

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Dec 12, 2012 (4 years and 11 months ago)

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Pre AP Biology

Chapter 16


Recombinant DNA and Genetic Engineering

I.

Introduction

1.

Gene therapy

is transfer of one or more normal or modified genes into an individual’s
body cells to correct genetic defect or boost resistance to disease

2.

Recombinant DNA technology has huge potential for medicine, agriculture, and industry

3.

By artificial selection practices, we produce new plants, breeds of cattle, cats, dogs, and
birds (meatier turkeys, sweeter oranges, seedless watermelon)

4.

We produce hyb
rids


mules (horse X donkey); tangelos (tangerine X grapefruit)

5.

Researchers analyze genes with
recombinant DNA technology
:

a.

Cut and recombine DNA from different species and insert it into bacterial, yeast, or
mammalian cells, which replicate their DNA and
divide rapidly

b.

Foreign DNA is copied are if it were their own and churn out useful quantities of
recombinant DNA molecules


basis of
genetic engineering


c.

Genes are isolated, modified, and inserted into the same organism or into a
different one; protein pr
oducts of modified gene may cover
function of missing or
malfunctioning counterparts

II.

16.1
A Toolkit for Making Recombinant DNA

1.

Restriction enzymes

a.

Enzymes that make cuts at specific sites along a DNA molecule

b.

recognize and cut specific sequences of four to

eight bases in DNA

c.

cuts are staggered, leaving single
-
stranded “tails” on the end of a DNA fragment
(Table 16.1)

d.

used to study the
genome



all of the DNA in a haploid number of chromosomes for
a species; human genome about 3.2 million base pairs long

2.

Mo
dification Enzymes

a.

DNA fragments with staggered cuts have “sticky” ends, which means the single
-
stranded tail can base
-
pair with complementary tail of other DNA fragments cut by
same restriction enzyme

b.

Mix DNA fragments cut by same restriction enzyme, and
the sticky ends of any two
fragments having complementary base sequences will base
-
pair and form a
recombinant DNA molecule (picture page 254)

c.

DNA ligase
, modification enzyme, seals the fragments

3.

Cloning Vectors for Amplifying DNA

a.

Restriction and
modification enzymes make it possible to insert foreign DNA into
bacterial cells

b.

Bacterial cell has one circular chromosome, may also inherit
plasmids



very small
circle of extra DNA that has just a few genes, gets replicated with bacterial
chromosome; ce
lls can survive without plasmids, but some genes offer benefits


antibiotic resistance

c.

Bacteria normally divide rapidly and often, huge populations of identical cells form

1.)

A cell can hold many identical copies of foreign DNA

2.)

In laboratories, foreign DNA

is inserted

into a plasmid for replication, becoming
a
DNA clone
, because bacterial cells have made many identical, “cloned” copies
of it

d.

Modified plasmid that accepts foreign DNA is a
cloning vector
; can be inserted into
a host bacterium, yeast to start
a “cloning factory”; a population of cells with
identical copies of the foreign DNA

4.

Reverse Transciptase to make cDNA

a.

We analyze genes to understand gene products and how they are put to use

b.

Most eukaryotic genes have introns (commercials), mRNA transcript
s cannot be
translated until introns snipped out and exons spliced together; bacterial cells can’t
snip out introns, can’t translate human genes into proteins

c.

cDNA



DNA strand copied from mRNA transcript;
reverse transcriptase

catalyzes
transcription in reverse, enzyme from RNA viruses build a complementary DNA
strand
on an mRNA transcript, other enzymes replace mRNA with complementary
DNA strand, forming double
-
stranded cDNA

d.

modified cDNA can be inserted into plasmid for ampli
fication, cells will synthesize
protein of interest

III.

16.2
PCR


A Faster Way to Amplify DNA

1.


Polymerase chain reaction (
PCR
)


way to amplify fragments of chromosomal DNA or
cDNA

2.

Primers
are synthetic, short nucleotide sequences, used to START PCR at the s
ite just
before the gene of interest

3.

PCR amplifies samples that contain even tiny amounts of DNA


forensics, research

IV.

16.3


DNA Fingerprinting

1.

No two people have exactly the same DNA base sequence, differences in sequences
distinguish one person from ano
ther

2.

Humans have unique sets of fingerprints; each human also has unique DNA fingerprint


unique array of DNA sequences inherited from parents, so accurate, can reveal
differences between full siblings

3.

More than 99% of DNA in all humans is exactly the sa
me; DNA fingerprinting focuses on
portion that tends to be highly variable from one person to next

a.

Human genome has tandem repeats


short DNA sequences present in many copies,
one after another, in a chromosome

b.

Individual with TTTTC repeated four times in one location, another individual might
have same repeat fifteen times in same location

4.

Researchers detect differences at tandem repeat sites with gel electrophoresis


uses
electric field to force molecules thr
ough a viscous gel

a.

Separates DNA fragments according to length


size dictates how far each fragment
will move through gel

b.

Tandem repeats of different sizes migrate at different rates

5.

Gel is immersed in buffer solution, DNA fragments from individual is loa
ded into gel

a.

Electric current is applied to solution, one end takes on an negative charge, the
other a positive charge

b.

Negatively charged phosphate of DNA fragment migrate through gel toward
positively charged pole

c.

Fragments move at different rates and sep
arate into bands according to length
;
smaller the fragment, faster it migrates; after set time, identify fragments of
different lengths

6.

DNA fingerprints help forensic scientists identify criminals, victims, and innocent
suspects; few drops of blood, semen,

or cells f
rom hair follicle, or skin cells

7.

DNA fingerprinting is firmly established as an accurate, unambiguous procedure,
routinely submitted as evidence in disputes of paternity; used to convict the guilty or
exonerate the innocent; PCR can be used to a
mplify the tandem
-
repeat region

V.

16.6 Using The Genetic Scripts

1.

Genetically engineered bacteria can produce medically valued proteins; huge bacterial
populations produce large quantities in stainless steel vats

a.

Diabetics


insulin

b.

Human somatotropin



huma
n growth hormone

c.

Blood
-
clotting factors

d.

Interferon


immune response, trigger body response to pathogens

e.

Vaccines

2.

Modified bacteria used in industry and cleaning up environmental messes

a.

Many microorganisms can break down organic wastes, cycle nutrients through
ecosystems

b.

Break down crude oil when sprayed on oil spills

c.

Take up excess phosphates or heavy metals from environment

3.

Bacterial species that contain plasmids offer benefits for b
asic research, agriculture, and
gene therapy

4.

Designing antibiotics and other defenses against new gene products


pathogens
undergo natural selection to evade a host organisms natural defenses

VI.

16.7 Designer Plants

1.

Researchers routinely regenerate crop plan
ts and many other plant species from
cultured cells

2.

Whole plants regenerated from preselected cultured cells can be hybridized with other
varieties of plants; desired genes might be transferred to plant linage to improve
herbicide resistance, pest resistan
ce, other traits that benefit crops and gardens

3.

Food supply for most of human population is vulnerable; farmers prefer genetically
similar varieties of high
-
yield plants, discard more diverse and old varieties

4.

Botanists collect seeds of wild ancestors of
potatoes, corn, other crop plants; send them
to seed banks


safe storage facilities designed to preserve genetic diversity




VII.

16.8
Gene Transfers in Animals

1.

Human gene transfers are being attempted in research into molecular basis of genetic
disorders

2.

Goa
ts produce CFTR protein (treat cystic fibrosis) and TPA (counter effects of heart
attack)

3.

Cattle may soon be producing human collagen, help repair cartilage, bone, and skin

4.

1990, Human Genome Initiative(Project) started

5.

Studies of genome of humans and ot
her organisms grouped together as a new research
field: genomics

a.

Structural genomics


concerned with actual mapping and sequencing of genomes
of individuals

b.

Comparative genomics


concerned with possible evolutionary relationships of
groups of organisms

c.

Genomics has potential for human gene therapy


inserting modified genes into
host cell of a particular tissue

VIII.

16.9 Safety Issues

1.

Do potential benefits of gene modifications and gene transfers outweigh potential
dangers?

2.

Genetically engineered bacteria
“designed” so cannot survive outside the laboratory

3.


“failsafe” genes built in the foreign DNA in case of escape


genes are silent unless
exposed to environmental conditions
, then get activated with lethal results

a.

Strawberry resistant to frost

b.

Potatoes t
oxic to insects that eat them

c.

Crops designed to resist weed killers

4.

If engineered plants or animals transfer modified gene to wild


weeds with resistance
would take over

5.

Standards for rigorous and extended safety tests are in place before modified organi
sms
enter environment