-Part

IV

-Chapter 20-part II

12/19/2014

DNA technology allows us to study the

sequence, expression, and function of a gene
DNA cloning allows researchers to
Compare genes and alleles between individuals
Locate gene expression in a body
Determine the role of a gene in an organism

Several techniques are used to analyze the DNA

of genes

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 nucleic acids or proteins by size
A current is applied that causes charged
molecules to move through the gel
Molecules are sorted into bands by their size

Fig. 20-9
TECHNIQUE
Mixture of
DNA molecules of
different
sizes

Power
source
Cathode

Anode +

Gel

Power
source

Longer
molecules

Shorter
molecules

RESULTS

 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
The procedure is also used to prepare pure
samples of individual fragments
5

Fig. 20-10

Normal -globin allele

175 bp

DdeI

Sickle-cell
allele

Large fragment

201 bp

DdeI

Normal
allele

DdeI

DdeI

Large
fragment

Sickle-cell mutant -globin allele

376 bp

DdeI

201 bp
175 bp

Large fragment

376 bp

DdeI

DdeI

(a) DdeI restriction sites in normal and

sickle-cell alleles of -globin gene

(b) Electrophoresis of restriction fragments

from normal and sickle-cell alleles

Southern Blotting ()
A technique called Southern blotting
combines gel electrophoresis of DNA
fragments with nucleic acid hybridization
Specific DNA fragments can be identified by
Southern blotting, using labeled probes that
hybridize to the DNA immobilized on a blot of
gel

Fig. 20-11
TECHNIQUE
DNA + restriction enzyme

Restriction
fragments

II

III

Heavy
weight

Nitrocellulose
membrane (blot)
Gel
Sponge

I Normal II Sickle-cell III Heterozygote

-globin allele
allele

2 Gel electrophoresis

1 Preparation of restriction fragments

Paper
towels

Alkaline
solution

3 DNA transfer (blotting)

Radioactively labeled
probe for -globin gene

II

III

Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele

Nitrocellulose blot

Fragment from
normal -globin
allele

4 Hybridization with radioactive probe

II

III

Film
over
blot
5 Probe detection

Introduction to Next Generation

Sequencing (NGS)
Sequencing History
1970s

Sanger Sequencing(1st Generation Sequencing)

By Frederick Sanger, Walter Gilbert, and Allan Maxam

1990

Human Genome Project (HGP)

Whole genome shotgun sequencing

2005

NGS(2nd Generation Sequencing)

2005

Roche 454 Pyrosequencing

2006

Illumina Solexa Genome Analyzer (GA)

2007

Applied Biosystems SOLiD sequencer

2008

Helicos Heliscope

2010

3rd Generation Sequencing

2010

Pacific Biosciences Single Molecule Real Time (SMRT)

2013

Oxford Nanopore Strand Sequencing

New Sequencing Technologies

Methods:
Pyrosequencing: 454 life sciences (Roche)
Sequencing by synthesis: Solexa (Illumina)
Sequencing by ligation: SOLiD (Sequencing by
Oligonucleotide Ligation and Detection) (ABI)

10

Illumina Sequencing Technology

1 Library

Preparation

2 Cluster

Generation

Attach DNA to flow cell

Perform bridge amplification
Generate clusters
Anneal sequencing primer

3 Sequencing

Extend first base, read, and

deblock
Repeat step above to extend
strand
Generate sequencing images

4 Data Analysis

http://www.illumina.com

Fragment DNA
Repair ends/Add A overhang
Ligate adapters
Select ligated DNA

Perform image analysis

Base calling
Demultiplexing/Generate FASTQ
Aligning
Detect variants and counting

11

NGS Comparisons1

Picture as taken from Kircher and Kelso, "High-throughput DNA sequencing concepts and limitations." Bioessays. Jun.
12
2010, pp.524-536.

NGS Comparisons2

Picture as taken from Kircher and Kelso, "High-throughput DNA sequencing concepts and limitations." Bioessays. Jun.
2010, pp.524-536.
13

Next Generation Sequencer454 Sequencing

James Watsons Genome

14

3rd Generation Sequencing

https://www.nanoporetech.com/technology/analytes-and-applications-dna-rna-proteins
/dna-an-introduction-to-nanopore-sequencing

15

Analyzing Gene Expression

Nucleic acid probes can hybridize with mRNAs
transcribed from a gene
Probes can be used to identify where or when
a gene is transcribed in an organism

16

Studying the Expression of Single Genes

Changes in the expression of a gene during
embryonic development can be tested using
Northern blotting ()
Reverse transcriptase-polymerase chain
reaction

Both methods are used to compare mRNA

from different developmental stages

17

 Northern blotting combines gel

electrophoresis of mRNA followed by
hybridization with a probe on a membrane
Identification of mRNA at a particular
developmental stage suggests protein function
at that stage

18

 Reverse transcriptase-polymerase chain

reaction (RT-PCR) is quicker and more
sensitive
Reverse transcriptase is added to mRNA to
make cDNA, which serves as a template for
PCR amplification of the gene of interest
The products are run on a gel and the mRNA
of interest identified

19

Fig. 20-13

TECHNIQUE
1 cDNA synthesis

mRNAs

cDNAs
2 PCR amplification

Primers

-globin
gene
3 Gel electrophoresis
RESULTS

Embryonic stages
1 2 3 4 5
6

20

Hybridization or high-throughput
sequencing?
Microarray(hybridization)
Advantage
Specific binding without
library size effect

Disadvantages
Rely on the existing knowledge
Cross hybridization
Platform differences

NGS(high-throughput)
Advantages
De novo analysis
High resolutions
Disadvantages
Affected by library size effect
Platform differences

21

Determining Gene Function

One way to determine function is to disable the
gene and observe the consequences
Using in vitro mutagenesis, mutations are
introduced into a cloned gene, altering or
destroying its function
When the mutated gene is returned to the cell,
the normal genes function might be
determined by examining the mutants
phenotype
22

 Gene expression can also be silenced using

RNA interference (RNAi)
Synthetic double-stranded RNA molecules
matching the sequence of a particular gene are
used to break down or block the genes mRNA

23

The Mechanism of RNAi

RISC: RNA-induced
silencing complex
24

Genetic Markers and Diseases

Single nucleotide polymorphisms (SNPs,
) are useful genetic markers
These are single base-pair sites that vary in a
population
When a restriction enzyme is added, SNPs result
in DNA fragments with different lengths, or
restriction fragment length polymorphism
(RFLP,)

Genetic disorders can be tested for using

genetic markers that are linked to the diseasecausing allele
25

Fig. 20-21

DNA
T
Normal allele
SNP
C
Disease-causing
allele

26

Cloning Animals: Nuclear Transplantation

()

In nuclear transplantation, the nucleus of an

unfertilized egg cell or zygote is replaced with
the nucleus of a differentiated cell
Experiments with frog embryos have shown
that a transplanted nucleus can often support
normal development of the egg
However, the older the donor nucleus, the
lower the percentage of normally developing
tadpoles

27

Reproductive Cloning of Mammals

In 1997, Scottish researchers announced the
birth of Dolly, a lamb cloned from an adult
sheep by nuclear transplantation from a
differentiated mammary cell
Dollys premature death in 2003, as well as her
arthritis, led to speculation that her cells were
not as healthy as those of a normal sheep,
possibly reflecting incomplete reprogramming
of the original transplanted nucleus
28

Fig. 20-18

TECHNIQUE
Mammary
cell donor

Egg cell
donor
2

Egg cell
from ovary
3 Cells fused
Cultured
mammary cells 3

4 Grown in

Nucleus
removed

Nucleus from
mammary cell

culture

Early embryo
5 Implanted

in uterus
of a third
sheep

Surrogate
mother

6 Embryonic

development

RESULTS

Lamb (Dolly)
genetically identical to
mammary cell donor

29

 Since 1997, cloning has been demonstrated in

many mammals, including mice, cats, cows,
horses, mules, pigs, and dogs
CC (for Carbon Copy) was the first cat cloned;
however, CC differed somewhat from her female
parent
Cloned animals do not always look or behave
exactly the same

30

Figure 20.20

31

Problems Associated with Animal Cloning

In most nuclear transplantation studies, only a
small percentage of cloned embryos have
developed normally to birth
Many epigenetic changes, such as acetylation
()of histones or methylation () of
DNA, must be reversed in the nucleus from a
donor animal in order for genes to be
expressed or repressed appropriately for early
stages of development
32

Stem Cells of Animals

A stem cell is a relatively unspecialized cell
that can reproduce itself indefinitely and
differentiate into specialized cells of one or
more types
Stem cells isolated from early embryos at the
blastocyst stage are called embryonic stem
cells (); these are able to
differentiate into all cell types
The adult body also has stem cells, which
replace nonreproducing specialized cells
33

Fig. 20-20
Embryonic stem cells

Adult stem cells

Early human embryo

at blastocyst stage
(mammalian equivalent of blastula)

From bone marrow

in this example

Cells generating
all embryonic
cell types

Cells generating
some cell types

Cultured
stem cells

Different
culture
conditions

Different
types of
differentiated
cells

Liver cells

Nerve cells

Blood cells

34

 Researchers can transform skin cells into ES

cells by using viruses to introduce stem cell
master regulatory genes
These transformed cells are called iPS cells
(induced pluripotent cells,)
These cells can be used to treat some diseases
and to replace nonfunctional tissues

35

36

Figure 20.22

1 Remove skin cells

from patient.

2 Reprogram skin cells

so the cells become
induced pluripotent
stem (iPS) cells.

Patient with
damaged heart
tissue or other
disease
3 Treat iPS cells so
that they differentiate
into a specific
cell type.
4 Return cells to
patient, where
they can repair
damaged tissue.

37

Human Gene Therapy ()

Gene therapy is the alteration of an afflicted
individuals genes
Gene therapy holds great potential for treating
disorders traceable to a single defective gene
Vectors are used for delivery of genes into
specific types of cells, for example bone marrow
Gene therapy raises ethical questions, such as
whether human germ-line cells should be
treated to correct the defect in future
generations
38

Fig. 20-22

Cloned
gene

Insert RNA version of normal allele

into retrovirus.

Viral RNA
2

Retrovirus
capsid

Let retrovirus infect bone marrow cells

that have been removed from the
patient and cultured.
3

Viral DNA carrying the normal

allele inserts into chromosome.

Bone
marrow
cell from
patient

Inject engineered
cells into patient.

Bone
marrow
39

Pharmaceutical Products
Advances in DNA technology and genetic
research are important to the development of
new drugs to treat diseases

40

Synthesis of Small Molecules for

Use as Drugs
The drug imatinib (Gleevec, receptor tyrosine
kinase inhibitor) is a small molecule that inhibits
overexpression of a specific leukemia-causing
receptor
Pharmaceutical products that are proteins can be
synthesized on a large scale

41

Protein Production in Cell Cultures

Host cells in culture can be engineered to
secrete a protein as it is made, simplifying the
task of purifying it
This is useful for the production of insulin,
human growth hormones, and vaccines

42

Protein Production by Pharm Animals

Transgenic animals are made by introducing
genes from one species into the genome of
another animal
Transgenic animals are pharmaceutical
factories, producers of large amounts of
otherwise rare substances for medical use

43

Figure 20.24

Secret a human blood protein, antithrombin, in goat milk

44

Forensic Evidence and Genetic Profiles

An individuals unique DNA sequence, or
genetic profile, can be obtained by analysis of
tissue or body fluids
Genetic profiles can be used to provide
evidence in criminal and paternity cases and to
identify human remains
Genetic profiles can be analyzed using RFLP
analysis by Southern blotting

45

 Even more sensitive is the use of genetic

markers called short tandem repeats (STRs,
), which are variations in the
number of repeats of specific DNA sequences
PCR and gel electrophoresis are used to
amplify and then identify STRs of different
lengths
The probability that two people who are not
identical twins have the same STR markers is
exceptionally small
46

Fig. 20-24

(a) This photo shows Earl

Washington just before
his release in 2001,
after 17 years in prison.

Source of
sample

STR
marker 1

STR
marker 2

STR
marker 3

Semen on victim

17, 19

13, 16

12, 12

Earl Washington

16, 18

14, 15

11, 12

Kenneth Tinsley

17, 19

13, 16

12, 12

(b) These and other STR data exonerated Washington and

led Tinsley to plead guilty to the murder.

47

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
Biofuels make use of crops such as corn,
soybeans, and cassava () to replace fossil
fuels

48

Agricultural Applications
DNA technology is being used to improve
agricultural productivity and food quality
Genetic engineering of transgenic animals
speeds up the selective breeding process
Beneficial genes can be transferred between
varieties of species

49

Safety and Ethical Questions Raised

by DNA Technology
Potential benefits of genetic engineering
must be weighed against potential hazards of
creating harmful products or procedures
Guidelines are in place in the United States
and other countries to ensure safe practices
for recombinant DNA technology

50

 Most public concern about possible hazards

centers on genetically modified (GM, )
organisms used as food
Some are concerned about the creation of
super weeds from the transfer of genes from
GM crops to their wild relatives
Other worries include the possibility that
transgenic protein products might cause allergic
reactions
51