1. DNA Technology & Genomics
Chapter 20

2. Biotechnology Today Genetic Engineering Our tool kit…
manipulation of DNA
if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with
this unit is a survey of those tools…
Our tool kit…

3. Understanding and Manipulating Genomes
DNA sequencing has depended on advances in technology, starting with making recombinant DNA
Recombinant DNA - nucleotide sequences from 2 different sources are combined in vitro into same DNA molecule
Methods for making recombinant DNA are central to genetic engineering, direct manipulation of genes for practical purposes

4. 20.1: DNA cloning permits production of multiple copies of a specific gene
To work with specific genes, scientists prepare gene-sized pieces of DNA in identical copies - gene cloning

5. Using Restriction Enzymes to Make Recombinant DNA
Bacterial restriction enzymes - cut DNA molecules at DNA sequences called restriction sites
Restriction enzyme usually makes many cuts, yielding restriction fragments
Most useful restriction enzymes cut DNA in a staggered way - producing fragments with “sticky ends” - bond with complementary “sticky ends” of other fragments
DNA ligase - enzyme that seals bonds between restriction fragments

6. 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

7. Cloning a Eukaryotic Gene in a Bacterial Plasmid
In gene cloning, original plasmid is called a cloning vector
Cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there

8. Producing Clones of Cells
Cloning human gene in bacterial plasmid:
1. Vector and gene-source DNA are isolated
2. DNA is inserted into 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

9. Cut both DNA samples with the same restriction enzyme. Human DNA
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

10. 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

11. 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

12. Identifying Clones Carrying a Gene of Interest
Clone carrying gene of interest can be identified with a nucleic acid probe
Called nucleic acid hybridization
Radioactive or fluorescent probes are engineered to be complimentary to a target sequence
First, denature of cells’ DNA

13. 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.

14. Storing Cloned Genes in DNA Libraries
Genomic library - collection of recombinant vector clones produced by cloning DNA fragments from an entire genome

15. Complementary DNA (cDNA) library - made by cloning DNA made in vitro by reverse transcription of all mRNA produced by a particular cell
cDNA library - represents only part of genome—only subset of genes transcribed into mRNA in original cells
Solves problem of prokaryotes not having machinery to remove introns

16. Bacterial Expression Systems
Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
Have to overcome differences in promoters and other DNA control sequences

17. Eukaryotic Cloning and Expression Systems
Use of 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 posttranslational modifications that many proteins require

18. Introducing recombinant DNA into eukaryotic cells:
electroporation, - applying a brief electrical pulse to create temporary holes in plasma membranes
inject DNA into cells using microscopic needles

19. Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) - produce many copies of specific target segment of DNA
3-step cycle: heating, cooling, and replication
chain reaction that produces an exponentially growing population of identical DNA molecules

20. 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

21. Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites
Restriction fragment analysis - detects differences in nucleotide sequences of DNA molecules
provide comparative information about DNA sequences

22. Gel Electrophoresis and Southern Blotting
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
Uses a gel as a molecular sieve to separate nuclei acids or proteins by size
DNA is negatively charged and moves towards a positive charge when placed in an electrical field

24. RFLP Analysis
restriction fragment analysis - fragments of DNA molecule are sorted by gel electrophoresis
Useful for comparing two different DNA molecules, such as two alleles for a gene

25. Restriction Fragment Length Differences as Genetic Markers
Restriction fragment length polymorphisms (RFLPs, or Rif-lips) - differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths
A RFLP can serve as genetic marker for a particular location (locus) in the genome
RFLPs are detected by Southern blotting

26. LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel
Sickle-cell mutant b-globin allele
376 bp
Large fragment
Ddel
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
-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

27. Uses: Evolutionary relationships
Comparing DNA samples from different organisms to measure evolutionary relationships
turtle
snake
rat
squirrel
fruitfly – 1 3 2 4 5 1 2 3 4 5
DNA  +

28. Uses: Medical diagnostic
Comparing normal allele to disease allele
chromosome with normal allele 1
chromosome with disease-causing allele 2
allele 1
allele 2 –
DNA 
Example: test for Huntington’s disease +

29. Uses: Forensics
Comparing DNA sample from crime scene with suspects & victim
suspects
crime scene sample S1 S2 S3 V –
DNA  +

30. DNA fingerprints
Comparing blood samples on defendant’s clothing to determine if it belongs to victim
DNA fingerprinting
comparing DNA banding pattern between different individuals
~unique patterns

31. Southern blotting - combines gel electrophoresis with nucleic acid hybridization
Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to DNA immobilized on a “blot” of gel

32. DNA + restriction enzyme
LE 20-10
Heavy
weight
DNA + restriction enzyme
Restriction
fragments I I
I
Nitrocellulose
paper (blot)
Gel
Sponge
Paper
towels
I Normal
-globin
allele
I Sickle-cell
allele
I Heterozygote
Alkaline
solution
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.
Probe hydrogen-
bonds to fragments
containing normal
or mutant -globin I I
I
Radioactively
labeled probe
for -globin
gene is added
to solution in
a plastic bag I I
I
Fragment from
sickle-cell
-globin allele
Film over
paper blot
Fragment from
normal -globin
allele
Paper blot
Hybridization with radioactive probe.
Autoradiography.

33. Concept 20.3: Entire genomes can be mapped at the DNA level
Most ambitious mapping project to date has been the sequencing of the human genome
Officially begun as Human Genome Project in 1990, sequencing was largely completed by 2003
Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

34. Genetic (Linkage) Mapping: Relative Ordering of Markers
1st stage is constructing linkage map of several thousand genetic markers throughout each chromosome
Order of markers and relative distances between them are based on recombination frequencies

35. LE 20-11 Cytogenetic map Chromosome bands Genes located by FISH
Genetic (linkage)
mapping
Genetic
markers
Physical mapping
Overlapping
fragments
DNA sequencing

36. Physical Mapping: Ordering DNA Fragments
Physical map - constructed by cutting DNA molecule into many short fragments and arranging them in order by identifying overlaps
Physical mapping gives actual distance in base pairs between markers

37. DNA Sequencing
Relatively short DNA fragments can be sequenced by dideoxy chain-termination method
Inclusion of special dideoxyribonucleotides in reaction mix ensures that fragments of various lengths will be synthesized

38. LE 20-12 DNA (template strand) Primer Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged) 3¢ 5¢ 5¢
DNA
polymerase 3¢
DNA (template
strand)
Labeled strands 5¢ 3¢ 3¢
Direction
of movement
of strands
Laser
Detector

39. Linkage mapping, physical mapping, and DNA sequencing represent overarching strategy of 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

40. 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

41. Concept 20.4: Genome sequences provide clues to important biological questions
In genomics, scientists study whole sets of genes and their interactions
Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

42. 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
NOVA Science Now: Public Genomes

44. Determining Gene Function
One way to determine function is to disable gene and observe consequences
Using in vitro mutagenesis, mutations are introduced into cloned gene, altering or destroying its function
When mutated gene is returned to cell, normal gene’s function might be determined by examining the mutant’s phenotype
In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes

45. 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

46. LE 20-14 Tissue sample Isolate mRNA. mRNA molecules
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.
Labeled cDNA molecules
(single strands)
DNA
microarray
Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.
Size of an actual
DNA microarray
with all the genes
of yeast (6,400 spots)

47. Comparing Genomes of Different Species
Comparative studies of genomes from related and widely divergent species provide information in many fields of biology
The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history
Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology
NOVA Science NOW: Autism Video

48. Future Directions in Genomics
Genomics - study of entire genomes
Proteomics - systematic study of all proteins encoded by a genome
Single nucleotide polymorphisms (SNPs) - provide markers for studying human genetic variation

49. Concept 20.5: The practical applications of DNA technology affect our lives in many ways
Many fields benefit from DNA technology and genetic engineering

50. Medical Applications
One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
Gene testing videohttp://

51. 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

52. RFLP marker Disease-causing allele Normal allele
DNA
Disease-causing
allele
Restriction
sites
Normal allele

53. 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

54. LE 20-16 Cloned gene 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.
Retrovirus
capsid
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Bone
marrow
Inject engineered
cells into patient.

55. Pharmaceutical Products
Some pharmaceutical applications of DNA technology:
Large-scale production of human hormones and other proteins with therapeutic uses
Production of safer vaccines

56. 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

57. Blood from defendant’s
LE 20-17
Defendant’s
blood (D)
Blood from defendant’s
clothes
Victim’s
blood (V)

58. 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

59. Agricultural Applications
DNA technology is being used to improve agricultural productivity and food quality

60. 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

62. 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

63. Agrobacterium tumefaciens
LE 20-19
Agrobacterium tumefaciens Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
Plant with
new trait

64. 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
Most public concern about possible hazards centers on genetically modified (GM) organisms used as food