1. DNA Technology

2. Sequencing of the human genome was largely completed by 2003
In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

3. An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

5. 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
Cloned genes are useful for making copies of a particular gene and producing a gene product

7. 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
Were discovered in bacteria to protect them from viruses dna takeover.
Methylation of cut site on bacterial DNA protects bacteria from getting cut up to
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

8. Restriction enzymes

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. How do you get DNA sequences of choice?
Bacterial DNA has not introns in its genome.
Need to create DNA without introns.
How do you do that.
Take mRNA and use reverse transcriptase to create DNA without introns.
Replicate that DNA to be inserted in Bacteria.

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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.

20. 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

21. 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

22. 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

23. 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

24. 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

26. Restriction fragment analysis detects DNA differences that affect restriction sites
Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules
Such analysis can rapidly provide comparative information about DNA sequences

27. 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 nuclei acids or proteins by size
Video: Biotechnology Lab

28. ELECTROPHORESIS

29. LE 20-8 Mixture of DNA Longer molecules molecules of differ- Cathode
ent sizes
Longer
molecules
Cathode
Shorter
molecules
Power
source
Gel
Glass
plates
Anode

31. 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

32. 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

33. 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.

34. Restriction Fragment Length Differences as Genetic Markers
Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths
A RFLP can serve as a genetic marker for a particular location (locus) in the genome
RFLPs are detected by Southern blottingGenetic (Linkage) Mapping: Relative Ordering of Markers
The first stage in mapping a large genome is constructing a linkage map of several thousand genetic markers throughout each chromosome
The 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
A physical map is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps
Physical mapping gives the actual distance in base pairs between markers

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

38. Sanger method of sequencing

39. 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

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

41. 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

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

44. 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

45. 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)

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

47. Medical Applications
One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

48. 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

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

50. 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

51. 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.

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

53. 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

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

55. 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

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

57. 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

59. 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

60. 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