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

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Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings



DNA Technology


Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


Sequencing of the human genome was largely
completed by 2003


In recombinant DNA, nucleotide sequences from
two different sources, often two species, are
combined
in vitro

into the same DNA molecule


Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


An example of DNA technology is the microarray,
a measurement of gene expression of thousands
of different genes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

DNA cloning permits production of multiple copies of a
specific gene or other DNA segment



To work directly with specific genes, scientists
prepare gene
-
sized pieces of DNA in identical
copies, a process called gene cloning

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

DNA Cloning and Its Applications:
A Preview


Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use
of bacteria and their plasmids


Cloned genes are useful for making copies of a
particular gene and producing a gene product

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Using Restriction Enzymes to Make Recombinant
DNA


Bacterial restriction enzymes cut DNA molecules at DNA
sequences called restriction sites


A restriction enzyme usually makes many cuts, yielding
restriction fragments


Were discovered in bacteria to protect them from viruses
dna takeover.


Methylation of cut site on bacterial DNA protects bacteria
from getting cut up to


The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky ends” that
bond with complementary “sticky ends” of other fragments


DNA ligase is an enzyme that seals the bonds between
restriction fragments

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Restriction enzymes


LE 20
-
3

Restriction site

DNA

5


3


3


5


Restriction enzyme cuts

the sugar
-
phosphate

backbones at each arrow.

One possible combination

DNA fragment from another

source is added. Base pairing

of sticky ends produces

various combinations.

Fragment from different

DNA molecule cut by the

same restriction enzyme

DNA ligase

seals the strands.

Recombinant DNA molecule

Sticky end

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Animation: Restriction Enzymes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

How do you get DNA sequences of choice?


Bacterial DNA has not introns in its genome.


Need to create DNA without introns.


How do you do that.


Take mRNA and use reverse transcriptase to
create DNA without introns.


Replicate that DNA to be inserted in Bacteria.

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Cloning a Eukaryotic Gene in a Bacterial Plasmid


In gene cloning, the original plasmid is called a
cloning vector


A cloning vector is a DNA molecule that can carry
foreign DNA into a cell and replicate there

LE 20
-
2

Bacterium

Bacterial

chromosome

Plasmid

Gene inserted into

plasmid

Cell containing gene

of interest

Gene of

interest

DNA of

chromosome

Recombinant

DNA (plasmid)

Plasmid put into

bacterial cell

Recombinant

bacterium

Host cell grown in culture

to form a clone of cells

containing the “cloned”

gene of interest

Protein expressed

by gene of interest

Protein harvested

Gene of

interest

Copies of gene

Basic

research

on gene

Basic

research

on protein

Basic research and

various applications

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up toxic waste

Protein dissolves

blood clots in heart

attack therapy

Human growth hor
-

mone treats stunted

growth

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Producing Clones of Cells


Cloning a human gene in a bacterial plasmid can
be divided into six steps:

1. Vector and gene
-
source DNA are isolated

2. DNA is inserted into the vector

3. Human DNA fragments are mixed with cut
plasmids, and base
-
pairing takes place

4. Recombinant plasmids are mixed with bacteria

5. The bacteria are plated and incubated

6. Cell clones with the right gene are identified

Animation: Cloning a Gene

LE 20
-
4_1

Isolate plasmid DNA

and human DNA.

Cut both DNA samples with

the same restriction enzyme.

Mix the DNAs; they join by base pairing.

The products are recombinant plasmids

and many nonrecombinant plasmids.

Bacterial cell

lac
Z gene

(lactose

breakdown)

Human

cell

Restriction

site

amp
R

gene

(ampicillin

resistance)

Bacterial

plasmid

Gene of

interest

Sticky

ends

Human DNA

fragments

Recombinant DNA plasmids

LE 20
-
4_2

Isolate plasmid DNA

and human DNA.

Cut both DNA samples with

the same restriction enzyme.

Mix the DNAs; they join by base pairing.

The products are recombinant plasmids

and many nonrecombinant plasmids.

Bacterial cell

lac
Z gene

(lactose

breakdown)

Human

cell

Restriction

site

amp
R

gene

(ampicillin

resistance)

Bacterial

plasmid

Gene of

interest

Sticky

ends

Human DNA

fragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cells

that have a mutation in their own
lacZ

gene.

Recombinant

bacteria

LE 20
-
4_3

Isolate plasmid DNA

and human DNA.

Cut both DNA samples with

the same restriction enzyme.

Mix the DNAs; they join by base pairing.

The products are recombinant plasmids

and many nonrecombinant plasmids.

Bacterial cell

lac
Z gene

(lactose

breakdown)

Human

cell

Restriction

site

amp
R

gene

(ampicillin

resistance)

Bacterial

plasmid

Gene of

interest

Sticky

ends

Human DNA

fragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cells

that have a mutation in their own
lacZ

gene.

Recombinant

bacteria

Plate the bacteria on agar

containing ampicillin and X
-
gal.

Incubate until colonies grow.

Colony carrying non
-

recombinant plasmid

with intact
lacZ

gene

Colony carrying

recombinant

plasmid with

disrupted
lacZ

gene

Bacterial

clone

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Identifying Clones Carrying a Gene of Interest


A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene


This process is called nucleic acid hybridization


An essential step in this process is denaturation of
the cells’ DNA, separation of its two strands

LE 20
-
5

Master plate

Filter

Solution

containing

probe

Filter lifted

and flipped over

Radioactive

single
-
stranded

DNA

Probe

DNA

Gene of

interest

Single
-
stranded

DNA from cell

Film

Hybridization

on filter

Master plate

Colonies

containing

gene of

interest

A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.

The filter is treated to break
open the cells and denature
their DNA; the resulting
single
-
stranded DNA
molecules are treated so that
they stick to the filter.

The filter is laid
under photographic
film, allowing any
radioactive areas to
expose the film
(autoradiography).

After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.

LE 20
-
6

Bacterial

clones

Recombinant

plasmids

Recombinant

phage DNA

or

Foreign genome

cut up with

restriction

enzyme

Phage

clones

Plasmid library

Phage library

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Bacterial Expression Systems


Several technical difficulties hinder expression of
cloned eukaryotic genes in bacterial host cells


To overcome differences in promoters and other
DNA control sequences, scientists usually employ
an expression vector, a cloning vector that
contains a highly active prokaryotic promoter

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


One method of introducing recombinant DNA into
eukaryotic cells is electroporation, applying a brief
electrical pulse to create temporary holes in
plasma membranes


Alternatively, scientists can inject DNA into cells
using microscopic needles


Once inside the cell, the DNA is incorporated into
the cell’s DNA by natural genetic recombination

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Amplifying DNA
in Vitro
: The Polymerase Chain
Reaction (PCR)


The polymerase chain reaction, PCR, can produce
many copies of a specific target segment of DNA


A three
-
step cycle

heating, cooling, and
replication

brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules

LE 20
-
7

Genomic DNA

Target

sequence

5


3


3


5


5


3


3


5


Primers

Denaturation:

Heat briefly

to separate DNA

strands

Annealing:

Cool to allow

primers to form

hydrogen bonds

with ends of

target sequence

Extension:

DNA polymerase

adds nucleotides to

the 3


end of eah

primer

Cycle 1

yields

2

molecules

New

nucleo
-

tides

Cycle 2

yields

4

molecules

Cycle 3

yields 8

molecules;

2 molecules

(in white boxes)

match target

sequence

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


http://www.dnalc.org/ddnalc/resources/pcr.html



http://spine.rutgers.edu/cellbio/flash/pcr.htm



http://highered.mcgraw
-
hill.com/olc/dl/120078/micro15.swf

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Restriction fragment analysis detects DNA
differences that affect restriction sites


Restriction fragment analysis detects differences
in the nucleotide sequences of DNA molecules


Such analysis can rapidly provide comparative
information about DNA sequences

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Gel Electrophoresis and Southern Blotting


One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis


This technique uses a gel as a molecular sieve to
separate nuclei acids or proteins by size

Video: Biotechnology Lab

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

ELECTROPHORESIS


http://www.dnalc.org/ddnalc/resources/electrophor
esis.html



http://www.sumanasinc.com/webcontent/animation
s/content/gelelectrophoresis.html


http://www.ndsu.nodak.edu/instruct/mcclean/plsc4
31/linkage/linkage6.htm



LE 20
-
8

Cathode

Power

source

Anode

Mixture

of DNA

molecules

of differ
-

ent sizes

Gel

Glass

plates

Longer

molecules

Shorter

molecules

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a
DNA molecule are sorted by gel electrophoresis


Restriction fragment analysis is useful for
comparing two different DNA molecules, such as
two alleles for a gene

LE 20
-
9

Normal
b
-
globin allele

175 bp

201 bp

Large fragment

Sikle
-
ell mutant
b
-
globin allele

376 bp

Large fragment

Dd
el

Dd
el

Dd
el

Dd
el

Dd
el

Dd
el

Dd
el

Dd
el restriction sites in normal and sickle
-
cell alleles of

b
-
globin gene

Normal

allele

Sickle
-
cell

allele

Large

fragment

376 bp

201 bp

175 bp

Electrophoresis of restriction fragments from normal

and sickle
-
cell alleles

LE 20
-
10

DNA + restriction enzyme

Restriction

fragments

I

Normal

b
-
globin

allele

I
I

Sikle
-
ell

allele

I
II

Heterozygote

Preparation of restriction fragments.

Gel electrophoresis.

Blotting.

I

I
I

II
I

Nitr潣ell畬潳e

paper (blot)

Gel

Sponge

Alkaline

solution

Paper

towels

Heavy

weight

Hybridization with radioactive probe.

I

I
I

II
I

Radioatively

labeled p牯re

for
b
-
globin

gene is added

to solution in

a plastic bag

Paper blot

Probe hydrogen
-

bonds to fragments

containing normal

or mutant
b
-
globin

Fragment from

sickle
-
cell

b
-
globin allele

Fragment from

normal
b
-
杬潢in

allele

Autoradiography.

I

I
I

II
I

䙩lm 潶er

灡灥r 扬潴

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Restriction Fragment Length Differences as
Genetic Markers


Restriction fragment length polymorphisms (RFLPs, or Rif
-
lips) are differences in DNA sequences on homologous
chromosomes that result in restriction fragments of
different lengths


A RFLP can serve as a genetic marker for a particular
location (locus) in the genome


RFLPs are detected by Southern blottingGenetic (Linkage)
Mapping: Relative Ordering of Markers


The first stage in mapping a large genome is constructing
a linkage map of several thousand genetic markers
throughout each chromosome


The order of markers and relative distances between them
are based on recombination frequencies

LE 20
-
11

Cytogenetic map

Genes located

by FISH

Chromosome

bands

Genetic

markers

Genetic (linkage)

mapping

Physical mapping

Overlapping

fragments

DNA sequencing

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Physical Mapping: Ordering DNA Fragments


A physical map is constructed by cutting a DNA
molecule into many short fragments and arranging
them in order by identifying overlaps


Physical mapping gives the actual distance in
base pairs between markers

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

DNA Sequencing


Relatively short DNA fragments can be sequenced
by the dideoxy chain
-
termination method


Inclusion of special dideoxyribonucleotides in the
reaction mix ensures that fragments of various
lengths will be synthesized

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Sanger method of sequencing

http://www.rvc.ac.uk/review/DNA_1/2_DNA_Seque
ncing.cfm

http://smcg.ccg.unam.mx/enp
-
unam/03
-
EstructuraDelGenoma/animaciones/secuencia.s
wf

http://www.dnalc.org/ddnalc/resources/sangerseq.
html

http://highered.mcgraw
-
hill.com/sites/0072556781/student_view0/chapte
r15/animation_quiz_1.html

LE 20
-
12

DNA

(template strand)

5


3


偲imer

3


5


DNA

polymerase

Deoxyribonucleotides

Dideoxyribonucleotides

(fluorescently tagged)

3


5


DNA (template

strand)

Labeled strands

3


Dire瑩on

of movement

of strands

Laser

Detector

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings


Linkage mapping, physical mapping, and DNA
sequencing represent the overarching strategy of
the Human Genome Project


An alternative approach to sequencing genomes
starts with sequencing random DNA fragments


Computer programs then assemble overlapping
short sequences into one continuous sequence

LE 20
-
13

Cut the DNA from
many copies of an
entire chromosome
into overlapping frag
-
ments short enough
for sequencing

Clone the fragments
in plasmid or phage

vectors

Sequence each fragment

Order the
sequences into one
overall sequence
with computer
software

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Identifying Protein
-
Coding Genes in DNA
Sequences


Computer analysis of genome sequences helps
identify sequences likely to encode proteins


The human genome contains about 25,000 genes,
but the number of human proteins is much larger


Comparison of sequences of “new” genes with
those of known genes in other species may help
identify new genes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Studying Expression of Interacting Groups of
Genes


Automation has allowed scientists to measure
expression of thousands of genes at one time
using DNA microarray assays


DNA microarray assays compare patterns of gene
expression in different tissues, at different times,
or under different conditions

LE 20
-
14

Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.

Apply the cDNA mixture to a
microarray, a microscope slide
on which copies of single
-
stranded DNA fragments from
the organism’s genes are fixed,
a different gene in each spot.
The cDNA hybridizes with any
complementary DNA on the
microarray.

Rinse off excess cDNA; scan
microarray for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.

Isolate mRNA.

Tissue sample

mRNA molecules

Labeled cDNA molecules

(single strands)

DNA

microarray

Size of an actual

DNA microarray

with all the genes

of yeast (6,400 spots)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Future Directions in Genomics


Genomics is the study of entire genomes


Proteomics is the systematic study of all proteins
encoded by a genome


Single nucleotide polymorphisms (SNPs) provide
markers for studying human genetic variation

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Medical Applications


One benefit of DNA technology is identification of
human genes in which mutation plays a role in
genetic diseases

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Diagnosis of Diseases


Scientists can diagnose many human genetic
disorders by using PCR and primers
corresponding to cloned disease genes, then
sequencing the amplified product to look for the
disease
-
causing mutation


Even when a disease gene has not been cloned,
presence of an abnormal allele can be diagnosed
if a closely linked RFLP marker has been found

LE 20
-
15

DNA

RFLP marker

Disease
-
causing

allele

Normal allele

Restriction

sites

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Human Gene Therapy


Gene therapy is the alteration of an afflicted
individual’s genes


Gene therapy holds great potential for treating
disorders traceable to a single defective gene


Vectors are used for delivery of genes into cells


Gene therapy raises ethical questions, such as
whether human germ
-
line cells should be treated
to correct the defect in future generations

LE 20
-
16

Cloned gene

Retrovirus

capsid

Bone

marrow

cell from

patient

Inject engineered

cells into patient.

Insert RNA version of normal allele

into retrovirus.

Viral RNA

Let retrovirus infect bone marrow cells

that have been removed from the

patient and cultured.

Viral DNA carrying the normal

allele inserts into chromosome.

Bone

marrow

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Pharmaceutical Products


Some pharmaceutical applications of DNA
technology:


Large
-
scale production of human hormones
and other proteins with therapeutic uses


Production of safer vaccines

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Forensic Evidence


DNA “fingerprints” obtained by analysis of tissue
or body fluids can provide evidence in criminal and
paternity cases


A DNA fingerprint is a specific pattern of bands of
RFLP markers on a gel


The probability that two people who are not
identical twins have the same DNA fingerprint is
very small


Exact probability depends on the number of
markers and their frequency in the population

LE 20
-
17

Defendant’s

blood (D)

Blood from defendant’s

clothes

Victim’s

blood (V)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Environmental Cleanup


Genetic engineering can be used to modify the
metabolism of microorganisms


Some modified microorganisms can be used to
extract minerals from the environment or degrade
potentially toxic waste materials

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Agricultural Applications


DNA technology is being used to improve
agricultural productivity and food quality

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Animal Husbandry and “Pharm” Animals


Transgenic organisms are made by introducing
genes from one species into the genome of
another organism


Transgenic animals may be created to exploit the
attributes of new genes (such as genes for faster
growth or larger muscles)


Other transgenic organisms are pharmaceutical
“factories,” producers of large amounts of
otherwise rare substances for medical use

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Genetic Engineering in Plants


Agricultural scientists have endowed a number of
crop plants with genes for desirable traits


The Ti plasmid is the most commonly used vector
for introducing new genes into plant cells

LE 20
-
19

Agrobacterium tumefaciens

Ti

plasmid

Site where

restriction

enzyme cuts

DNA with

the gene

of interest

T DNA

Recombinant

Ti plasmid

Plant with

new trait