Studying and Manipulating Genes


12 déc. 2012 (il y a 9 années et 3 mois)

490 vue(s)

Albia Dugger • Miami Dade College

Chapter 15

Studying and
Manipulating Genes

15.1 Personal DNA Testing

About 99% of your DNA is the same as everyone else


A nucleotide difference found in at least 1% of a population is
called a
nucleotide polymorphism (SNP)

SNPs account for many differences in the way humans look,
and in the way our bodies work

Personal DNA Testing

About 4.5 million SNPs in human DNA have been identified

Using commercial SNP chips, individuals can now be tested
for some of the SNPs they carry

Personal genetic testing may soon allow physicians to
customize treatments based on individual genetic makeup

Personal DNA Testing: SNP Chips

Cutting and Pasting DNA

Restriction enzymes

Bacterial enzymes that cut DNA wherever a specific
nucleotide sequence occurs

stranded DNA tails produced by the same restriction
enzyme base
pair together

DNA ligase bonds

sticky ends


Recombinant DNA

Composed of DNA from two or more organisms

Making Recombinant DNA

A restriction enzyme
recognizes a specific
base sequence (orange
boxes) in DNA from any


enzyme (cut)

The enzyme cuts
DNA from two sources
into fragments. This
enzyme leaves sticky



When the DNA
fragments from the

two sources are mixed
together, matching
sticky ends base
with each other.


DNA ligase


DNA ligase joins
the base
paired DNA
fragments. Molecules
of recombinant DNA
are the result.


Stepped Art

DNA Cloning

Making recombinant DNA is the first step in
DNA cloning
, a
set of laboratory methods that uses living cells to mass
produce specific DNA fragments

DNA cut into fragments by restriction enzymes is inserted into
cloning vectors

(plasmids) cut with the same enzyme

Cloning vectors with foreign DNA are placed in host cells,
which divide and produce many
, each with a copy of
the foreign DNA

Figure 15
3 p236

pDrive Cloning Vector

3.85 kb














DNA Cloning




A restriction enzyme cuts

a specific base sequence in
chromosomal DNA and in

a plasmid cloning vector.




DNA fragments


A fragment of chromosomal
DNA and the plasmid base
at their sticky ends. DNA ligase
joins the two pieces of DNA.

Stepped Art

The recombinant plasmid is
inserted into a host cell. When the
cell multiplies, it makes multiple
copies of the plasmids.


cDNA Cloning

Complementary DNA (cDNA)

DNA made from an mRNA template

Reverse transcriptase

transcribes mRNA to DNA, forming a
hybrid molecule

DNA polymerase builds a double
stranded DNA molecule
that can be cloned

cDNA Cloning

RI recognition site






Home Message:

What is DNA cloning?

DNA cloning uses living cells to mass
produce particular DNA
fragments; restriction enzymes cut DNA into fragments, then
DNA ligase seals the fragments into cloning vectors;
recombinant DNA molecules result

A cloning vector that holds foreign DNA can be introduced
into a living cell; when the host cell divides, it gives rise to
huge populations of genetically identical cells (clones), each
of which contains a copy of the foreign DNA

DNA Libraries


The entire set of genetic material of an organism

DNA libraries

are sets of cells containing various cloned
DNA fragments

Genomic libraries (all DNA in a genome)

cDNA libraries (all active genes in a cell)



A fragment of DNA labeled with a tracer

Used to find a specific clone carrying DNA of interest in a
library of many clones

Nucleic acid hybridization

Base pairing between DNA from different sources

A probe hybridizes with the targeted gene

Figure 15
5 p238

D A probe is added to the liquid bathing the paper. The probe hybridizes
pairs) with the spots of DNA that contain complementary base

C The paper is soaked in a solution that ruptures the cells and releases
their DNA. The DNA clings to the paper in spots mirroring the distribution
of colonies.

B A piece of special paper pressed onto the surface of the growth
medium will bind some cells from each colony.

A Individual bacterial cells from a DNA library are spread over the
surface of a solid growth medium. The cells divide repeatedly and form

clusters of millions of genetically identical descendant cells.

E The bound probe makes a spot. Here, one radioactive spot darkens x
ray film. The
position of the spot is compared to the positions of the original bacterial colonies. Cells
from the colony that made the spot are cultured, and the DNA they contain is harvested.


Polymerase chain reaction (PCR)

A cycled reaction that uses a heat
tolerant form of DNA
polymerase (

polymerase) to produce billions of copies
of a DNA fragment

PCR Techniques

DNA to be copied is mixed with DNA polymerase, nucleotides

that base
pair with certain DNA sequences

Cycles of high and low temperatures break and reform
hydrogen bonds between DNA strands, doubling the amount
of DNA in each cycle

Two Rounds of PCR

Home Message
: How do researchers
study one gene in the context of many?

Researchers isolate one gene from the many other genes in a
genome by making DNA libraries or with PCR

Probes are used to identify one clone that hosts a DNA
fragment of interest among many other clones in a DNA

PCR quickly mass
produces copies of a particular section of

DNA Sequencing

DNA is synthesized with normal nucleotides and
dideoxynucleotides tagged with different colors

When a tagged base is added, DNA synthesis stops;
fragments of all lengths are made


separates the fragments of DNA, each
ending with a tagged base, by length

The order of colored bases is the sequence of DNA

Figure 15
7a p240

DNA template





The Human Genome Project

Automated DNA sequencing and PCR allowed human
genome projects to sequence the 3 billion bases in the human

28,976 genes have been identified, but not all of their
products or functions are known

15.5 Genomics

Comparing the sequence of our genome with that of other
species is giving us insights into how the human body works

Unique sequences of genomic DNA can be used to
distinguish an individual from all others

Genomic DNA Alignment

DNA Chips

DNA chips

Microarrays of many different DNA samples arranged on a
glass plate

Used to compare patterns of gene expression among cells
of different types or under different conditions

May be used to screen for genetic abnormalities,
pathogens, or cancer

DNA Profiling: SNPs

Identifying an individual by his or her unique array of DNA
sequences is called
DNA profiling

One type of DNA profiling involves SNP
chips with
microscopic spots of DNA stamped on them

An individual

s genomic DNA hybridizes only with DNA spots
that have a matching SNP sequence

Probes reveal where the genomic DNA has hybridized

DNA Profiling: STRs

Another method of DNA profiling involves analysis of short
tandem repeats, sections of DNA in which a series of 4 or 5
nucleotides is repeated several times in a row.

Types and numbers of STRs vary greatly among individuals

Unless two people are identical twins, the chance that they
have identical short tandem repeats in even three regions of
DNA is 1 in a quintillion (10

Analyzing STRs

PCR is used to amplify DNA from regions of several
chromosomes that have STRs

Electrophoresis is used to separate the fragments and create
a unique DNA fingerprint

DNA fingerprints have many applications, including legal
cases, forensics, and population studies

An STR Profile

15.6 Genetic Engineering

Genetic engineering
is a laboratory process by which
deliberate changes are introduced into an individual


A gene may be altered and reinserted into an individual of the
same species, or a gene from one species may be transferred
to another to produce an organism that is transgenic

Both result in a genetically modified organism (GMO)

Some GMOs

The most common GMOs are bacteria and yeast


E. coli
have been modified to produce a fluorescent
protein from jellyfish

used to study gene expression

Some bacteria have been modified to produce medically
important proteins such as human insulin and chymotrypsin

Other GMO
produced enzymes improve the taste and clarity
of beer and fruit juice, slow bread staling, or modify fats

Home Message:

What is genetic engineering?

Genetic engineering is the deliberate alteration of an

s genome, and it results in a genetically modified
organism (GMO).

A transgenic organism carries a gene from a different
species; transgenic bacteria and yeast are used in research,
medicine, and industry

15.7 Designer Plants

Genetically engineered crop plants are widespread in the
United States

Introducing New Genes into Plants

Foreign or modified genes can be introduced into plant cells
by way of electric or chemical shocks, or by blasting them
with DNA
coated micropellets

Genes can also be introduced biologically using the Ti
plasmid, a plasmid of the bacterium

which contains tumor
inducing (Ti) genes

GMO Controversies

Many people are opposed to any GMO

The USDA Animal and Plant Health Inspection Service
(APHIS) has approved 78 GMO crop plants for use in US,
including corn, sorghum, cotton, soy, canola, alfalfa

In crops engineered for herbicide resistance, weeds are
becoming resistant to herbicides

Pollen containing engineered genes is spreading into wild
plants and nonengineered crops

Corn with the


Home Message:
Are genetically modified
plants used as commercial crops?

Genetically modified crop plants can help farmers be more
productive while reducing overall costs

The widespread use of GMO crops has had unintended
environmental effects

Herbicide resistant weeds are now common, and recombinant
genes have spread to wild plants and non
GMO crops

Some Genetically Modified Animals

Knockouts and Organ Factories

Transgenic pigs with human proteins that are not rejected by
the human immune system are a potential source of organs
and tissues for transplants in humans

Transplantation of a tissue or organ from one species to
another is called

Critics are concerned that pig
human transplants would
allow pig viruses to cross the species barrier and infect
humans, perhaps with catastrophic results

Gene Therapy

Gene therapy is now being tested as a treatment for several
types of cancer, sickle
cell anemia, cystic fibrosis, and other
inherited diseases

Gene therapy

Transfer of recombinant DNA into body cells to correct a
genetic defect or treat a disease

Viral vectors or lipid clusters insert an unmutated gene into
an individual

s chromosomes

Getting Worse

No one can predict where a virus
injected gene will insert into
a chromosome

Five of the 20 boys treated with gene therapy for SCID
developed cancer (leukemia), and one of them has died

In other studies, severe allergic reactions to the viral vector
itself have resulted in death

Getting Perfect


the selection of human being with the most
desirable traits

has been used as a justification for horrific
acts such as genocide

It may soon be possible to engineer humans for particular
desirable traits such as height, strength, or intelligence

Many questions must be answered about the ethics and
consequences of manipulating the human genome

Home Message:

Can people be genetically modified?

Genes can be transferred into a person

s cells to correct a
genetic defect or treat a disease

However, the outcome of altering a person

s genome
remains unpredictable given our current understanding of how
the genome works