Studying and Manipulating Genes

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Dec 12, 2012 (4 years and 7 months ago)

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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

s



A nucleotide difference found in at least 1% of a population is
called a
single
-
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



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


DNA ligase bonds

sticky ends


together



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
source.

1

restriction
enzyme (cut)


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

2

mix


When the DNA
fragments from the

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

3

DNA ligase

(paste)


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

4

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
clones
, each with a copy of
the foreign DNA

Figure 15
-
3 p236

pDrive Cloning Vector

3.85 kb

Kpn
l
Sph

l
Pst

l
Bam

Hl
Eco

RI
Sal

l
Acc

l
Xho

l
Xba

l
Bst

XI
Sac

l

Not

l

DNA Cloning

recombinant
plasmid

plasmid
cloning
vector

chromosomal
DNA

A
A restriction enzyme cuts

a specific base sequence in
chromosomal DNA and in

a plasmid cloning vector.

cut

plasmid

chromosomal

DNA fragments

B

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

Stepped Art

C
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

Eco
RI recognition site

cDNA

mRNA

cDNA

mRNA

DNA

Take
-
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





Genome


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)

Probes





Probe


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
(base
-
pairs) with the spots of DNA that contain complementary base
sequences.

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
colonies

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.

PCR






Polymerase chain reaction (PCR)


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

polymerase) to produce billions of copies
of a DNA fragment


PCR Techniques






DNA to be copied is mixed with DNA polymerase, nucleotides
and
primers

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

Take
-
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
library



PCR quickly mass
-
produces copies of a particular section of
DNA

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



Electrophoresis

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

2

3

4

5

The Human Genome Project






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



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
18
)

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

s
genome



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



Some

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


Take
-
Home Message:

What is genetic engineering?





Genetic engineering is the deliberate alteration of an
individual

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
Agrobacterium
tumefaciens

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
Bt

Gene

Take
-
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
xenotransplantation



Critics are concerned that pig
-
to
-
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
-
X1
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




Eugenics



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

Take
-
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