1. DNA Technology and Genomics
Chapter 20
DNA Technology and Genomics

2. Overview: Understanding and Manipulating Genomes
Sequencing of the human genome was largely completed by 2003
DNA sequencing has depended on advances in technology, starting with making recombinant DNA
In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule
Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

3. DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products
An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

5. Concept 20.1: 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

6. 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
For cloning genes, a plasmid is first isolated from a bacterial cell, then the foreign DNA is inserted into it
Results in recombinant DNA molecule
Cloned genes are useful for making copies of a particular gene and producing a gene product

7. LE 20-2 Bacterium Cell containing gene of interest Gene inserted into
plasmid
Bacterial
chromosome
Plasmid
Gene of
interest
Recombinant
DNA (plasmid)
DNA of
chromosome
Plasmid put into
bacterial cell
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Protein harvested
Basic research and
various applications
Basic
research
on gene
Basic
research
on protein
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

8. 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
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

9. One possible combination Recombinant DNA molecule
Restriction site
DNA 5¢ 3¢ 3¢ 5¢
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
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
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule

10. Animation: Restriction Enzymes

11. 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

12. 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

13. LE 20-4_1
Bacterial cell
lacZ gene
(lactose
breakdown)
Human
cell
Isolate plasmid DNA
and human DNA.
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Cut both DNA samples with
the same restriction enzyme.
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids

14. LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell
Isolate plasmid DNA
and human DNA.
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Cut both DNA samples with
the same restriction enzyme.
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own lacZ
gene.
Recombinant
bacteria

15. LE 20-4_3 Human DNA fragments Bacterial cell lacZ gene (lactose
breakdown)
Human
cell
Isolate plasmid DNA
and human DNA.
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Cut both DNA samples with
the same restriction enzyme.
Human DNA
fragments
Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
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

16. 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

17. LE 20-5 Colonies containing gene of interest Master plate Probe DNA
Radioactive
single-stranded
DNA
Solution
containing
probe
Gene of
interest
Filter
Single-stranded
DNA from cell
Film
Filter lifted
and flipped over
Hybridization
on filter
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.

18. Storing Cloned Genes in DNA Libraries
A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
A genomic library that is made using bacteriophages is stored as a collection of phage clones

19. Foreign genome cut up with restriction enzyme or Bacterial clones
LE 20-6
Foreign genome
cut up with
restriction
enzyme or
Bacterial
clones
Recombinant
plasmids
Phage
clones
Recombinant
phage DNA
Plasmid library
Phage library

20. A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell
A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

21. Cloning and Expressing Eukaryotic Genes
As an alternative to screening a DNA library, clones can sometimes be screened for a desired gene based on detection of its encoded protein
After a gene has been cloned, its protein product can be produced in larger amounts for research

22. 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

23. Eukaryotic Cloning and Expression Systems
The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems
YACs behave normally in mitosis and can carry more DNA than a plasmid
Eukaryotic hosts can provide the posttranslational modifications that many proteins require

24. 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

25. 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

26. LE 20-7 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ Denaturation:
Heat briefly
to separate DNA
strands 5¢ 3¢ 3¢ 5¢
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Cycle 1
yields 2
molecules
Primers
Extension:
DNA polymerase
adds nucleotides to
the 3¢ end of each
primer
New
nucleo-
tides
Cycle 2
yields 4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence