12.1 Genes can be cloned in recombinant plasmids

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GENE CLONING

Copyright © 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids


Genetic engineering

involves manipulating
genes for practical purposes


Gene cloning

leads to the production of multiple
identical copies of a gene
-
carrying piece of DNA


Recombinant DNA

is formed by joining DNA
sequences from two different sources


One source contains the gene that will be cloned


Another source is a gene carrier, called a vector


Plasmids

(small, circular DNA molecules
independent of the bacterial chromosome) are often
used as vectors


Copyright © 2009 Pearson Education, Inc.


Steps in cloning a gene

1.
Plasmid DNA is isolated

2.
DNA containing the gene of interest is isolated

3.
Plasmid DNA is treated with restriction enzyme that
cuts in one place, opening the circle

4.
DNA with the target gene is treated with the same
enzyme and many fragments are produced

5.
Plasmid and target DNA are mixed and associate with
each other

Copyright © 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids

6.
Recombinant DNA molecules are produced when
DNA ligase

joins plasmid and target segments
together

7.
The recombinant DNA is taken up by a bacterial cell

8.
The bacterial cell reproduces to form a
clone

of
cells

Copyright © 2009 Pearson Education, Inc.

12.1 Genes can be cloned in recombinant plasmids

Examples of

gene use

Recombinant

DNA

plasmid

E. coli

bacterium

Plasmid

Bacterial

chromosome

Gene of interest

DNA

Gene

of interest

Cell with DNA

containing gene

of interest

Recombinant

bacterium

Clone

of cells

Genes may be inserted

into other organisms

Genes or proteins

are isolated from the

cloned bacterium

Harvested

proteins

may be

used directly

Examples of

protein use

Gene

of interest

Isolate

plasmid

1

Isolate

DNA

2

Cut plasmid

with enzyme

3

Cut cell’s DNA

with same enzyme

4

Combine targeted fragment

and plasmid DNA

5

Add DNA ligase,

which closes

the circle with

covalent bonds

6

Put plasmid

into bacterium

by transformation

7

Allow bacterium

to reproduce

8

9

12.2 Enzymes are used to “cut and paste” DNA


Restriction enzymes

cut DNA at specific
sequences


Each enzyme binds to DNA at a different
restriction
site


Many restriction enzymes make staggered cuts that
produce
restriction fragments

with single
-
stranded
ends called “sticky ends”


Fragments with complementary sticky ends can
associate with each other, forming recombinant DNA


DNA fragments joined
together

Copyright © 2009 Pearson Education, Inc.

Restriction enzyme

recognition sequence

1

2

DNA

Restriction enzyme

cuts the DNA into

fragments

Sticky end

Restriction enzyme

recognition sequence

1

2

DNA

Restriction enzyme

cuts the DNA into

fragments

Sticky end

3

Addition of a DNA

fragment from

another source

Restriction enzyme

recognition sequence

1

2

DNA

Restriction enzyme

cuts the DNA into

fragments

Sticky end

3

Addition of a DNA

fragment from

another source

4

Two (or more)

fragments stick

together by

base
-
pairing

Restriction enzyme

recognition sequence

1

2

DNA

Restriction enzyme

cuts the DNA into

fragments

Sticky end

3

Addition of a DNA

fragment from

another source

4

Two (or more)

fragments stick

together by

base
-
pairing

DNA ligase

pastes the strands

Recombinant

DNA molecule

5

GENETICALLY MODIFIED
ORGANISMS

These are organisms that have had
foreign genes inserted into their
genome through plasmids created by
humans

Copyright © 2009 Pearson Education, Inc.

12.6 Recombinant cells and organisms can

mass
-
produce gene products


Cells and organisms containing cloned genes are
used to manufacture large quantities of gene
products


Remember how long it took us to create mRNA
and the protein hemoglobin


A cell can do this at a rate of 50
nts
/sec


Capabilities of the host cell are matched to the
characteristics of the desired
product

Copyright © 2009 Pearson Education, Inc.

12.6 Recombinant cells and organisms can

mass
-
produce gene products


Prokaryotic
host:
E. coli


Can
be engineered to secrete
proteins


Eukaryotic hosts


Yeast:
S.
cerevisiae


Can produce and secrete complex eukaryotic
proteins


Mammalian cells in culture


Can attach sugars to form
glycoproteins



Pharm
” animals


Will secrete gene product in milk


Copyright © 2009 Pearson Education, Inc.

How will this technology help humans?


What are some products that would be difficult for us to
synthesize but easy for a genetically engineered organism to
produce?

Copyright © 2009 Pearson Education, Inc.

How will this technology help humans?


What are some products that would be difficult for us to
synthesize but easy for a genetically engineered organism to
produce?

Copyright © 2009 Pearson Education, Inc.

Product

Use

Insulin

Diabetes

Growth hormones

Growth defects, burns,
weight gain in cattle

Il
-
2

Cancer

Taxol

Ovarian cancer

Hepatitis

B vaccine

Hepatitis B vaccine

TPA

Heart attacks and strokes

Pros

Cons

Genetic Modification of Organisms

Pros

Cons

Food that can deliver vaccines
-

bananas that
produce hepatitis B vaccine

Potential human health impact: allergens, transfer of
antibiotic resistance markers, unknown effects

More nutritious foods
-

rice with increased iron and
vitamins


Potential environmental impact: unintended transfer
of
transgenes

through cross
-
pollination, unknown
effects on other organisms (e.g., soil microbes), and
loss of flora and fauna biodiversity

Faster growing fish, fruit and nut trees

Domination of world food production by a few
companies

Drought resistant

Increasing dependence on
industralized

nations by
developing countries

Resistant to herbicides and pests

Violation of natural organisms’ intrinsic values

Mass produce proteins such as insulin

Tampering with nature by mixing genes among
species

Clean up the environment

Objections to consuming animal genes in plants and
vice versa

Stress for animal

Labeling not mandatory in some countries (e.g.,
United States)


Genetic Modification of Organisms

Real Life CSI

Copyright © 2009 Pearson Education, Inc.

Not always like you see on TV

Copyright © 2009 Pearson Education, Inc.

"We asked medical students 'Where did you get
some of your ideas before you even came into
the medical profession?' And interestingly
enough, 'ER came up as the number one
influence,"
Brindley

told Canada AM.


Knowing that ER was such an influence on the
students,
Brindley

and Needham decided to
watch a few episodes of the show and observe
their techniques.


"We watched two seasons of ER and not once
was the resuscitation done properly,"
Brindley

notes. "And that's despite having numerous
medical experts advising the show."


Real doctors influenced by TV dramas,
study suggests

March 26,2009


How do real detectives find out
who did it or who didn’t do it?

Copyright © 2009 Pearson Education, Inc.

DNA PROFILING

Copyright © 2009 Pearson Education, Inc.

12.11 The analysis of genetic markers can
produce a DNA profile


DNA profiling

is the analysis of DNA fragments
to determine whether they come from a particular
individual


Compares genetic markers from noncoding regions
that show variation between individuals


Involves amplification (copying) of markers for
analysis


Sizes of amplified fragments are compared

Copyright © 2009 Pearson Education, Inc.

Crime scene

DNA isolated

1

Suspect 1

Suspect 2

DNA of selected

markers amplified

2

Amplified DNA

compared

3

12.13 Gel electrophoresis sorts DNA molecules
by size


Gel electrophoresis

separates DNA molecules
based on size


DNA sample is placed at one end of a porous gel


Current is applied and DNA molecules move from the
negative electrode toward the positive electrode


Shorter DNA fragments move through the gel pores
more quickly and travel farther through the gel


DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence


Each band is a collection of DNA molecules of the
same length

Copyright © 2009 Pearson Education, Inc.

Video: Biotechnology Lab

Mixture of DNA

fragments of

different sizes

Completed gel

Longer

(slower)

molecules

Gel

Power

source

Shorter

(faster)

molecules

Gilbert Alejandro (Uvalde County, Texas)

Factual background. On the evening of April 27, 1990, a woman in her fifties came home and was
attacked from behind by a man. The man placed a pillow over her head and sexually assaulted her.
He then fled the house. The woman could not describe the man except for basic physical size. She
also noted that the man was wearing some kind of cap, a gray T
-
shirt, and dark
-
colored shorts. The
police canvassed the area and questioned three men, one of whom was wearing clothes matching the
victim's description. The police did not detain them. The victim picked out Alejandro from his
photograph in a mug book.


In October 1990 Gilbert Alejandro was convicted of aggravated sexual assault by a Uvalde County
jury. He was sentenced to 12 years in prison.


Prosecutor's evidence at trial. The prosecution based its case on several points:


The victim identified Alejandro from a police mug shot.


The victim identified Alejandro in court (although she stated that she had a pillow over her head
during the assault).


Fred
Zain
, the chief forensic expert for
BexarCounty
, Texas, testified that a DNA test of Alejandro's
sample matched DNA found on the victim's clothing "and could only have originated from him
[Alejandro]."


Alejandro's only alibi was from his mother, who testified that he was at home at the time of the
assault.


Postconviction

challenges



Bexar County performed the forensic laboratory work in this case for the Uvalde County prosecutor's office. Bexar
County discovered that the State's forensic expert in this case, Fred
Zain

(see also the Gerald Wayne Davis, William
O'Dell Harris, and Glen Woodall cases), had falsified results and lied about his credentials

when he was employed as a State police serologist in West Virginia. When Alejandro's lawyers were informed of this,
they filed a writ of habeas corpus. At this time, Alejandro was released to his parents and placed on electronic
monitoring.


On July 26, 1994, a Uvalde County District Court heard Alejandro's petition. Present at this hearing were an original
trial juror, the original jury foreman, and a Bexar County forensic DNA analyst. The two jurors testified that they
based their

guilty verdict solely on
Zain's

testimony and without his testimony the jury would have acquitted on the basis of
reasonable doubt. The DNA analyst testified that results from at least one other DNA test had excluded Alejandro. He
also testified that

the test to which
Zain

testified was inconclusive and could not have been the basis of a conviction.


DNA results. In July 1990 the original DNA tests done in this case

the ones
Zain

testified were
inculpatory


were


inconclusive. A Restriction Fragment Length Polymorphism (RFLP) test performed by the Bexar County crime
laboratory on October 3, 1990, excluded Alejandro as the source of the semen left on the victim's nightgown. The
district court


also reported that an additional test was done on December 19, 1990, after the trial, and it too
excluded Alejandro. According to the district court's findings of fact, Fred
Zain

knew of these exculpatory results and
failed to report them to anyone.


Conclusion. As a result of the findings of fact by the district court, the court of criminal appeals overturned Alejandro's
conviction and released him to stand trial again without
Zain's

testimony. The district attorney, however, declined to
prosecute the case. On September 21, 1994, Alejandro was released from electronic monitoring and all charges

were dismissed. Alejandro served 4 years of his sentence. On June 27, 1995, he was awarded $250,000 in a civil suit
against Bexar

12.14 STR analysis is commonly used for DNA
profiling


Short tandem repeats

(
STRs
) are genetic
markers used in DNA profiling


STRs are short DNA sequences that are repeated
many times in a row at the same location


The number of repeating units can differ between
individuals


STR analysis

compares the lengths of STR
sequences at specific regions of the genome


Current standard for DNA profiling is to analyze 13
different STR sites

Copyright © 2009 Pearson Education, Inc.

STR site 1

Crime scene DNA

STR site 2

Suspect’s DNA

Number of short tandem

repeats match

Number of short tandem

repeats do not match

12.16 RFLPs can be used to detect differences in
DNA sequences


Single nucleotide polymorphism

(
SNP
) is a
variation at one base pair within a coding or
noncoding sequence


Restriction fragment length polymorphism

(
RFLP
) is a variation in the size of DNA fragments
due to a SNP that alters a restriction site


RFLP analysis involves comparison of sizes of
restriction fragments by gel electrophoresis

Copyright © 2009 Pearson Education, Inc.

Crime scene

DNA

Suspect’s

DNA

Pedigree ex