1. Recombinant DNA Technology

2. This achievement ushered in the era of gene cloning
Recombinant DNA technology is the use of in vitro molecular techniques to isolate and manipulate fragments of DNA
In the early 1970s, researchers at Stanford University were able to construct chimeric molecules called recombinant DNA molecules
Shortly thereafter, it became possible to introduce such molecules into living cells where they replicated to make many identical copies.
This achievement ushered in the era of gene cloning
Recombinant DNA technology and gene cloning have been fundamental to our understanding of gene structure and function
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3. Cloning Experiments Involve Chromosomal and Vector DNA
Cloning experiments usually involve two kinds of DNA molecules
Chromosomal DNA
Serves as the source of the DNA segment of interest
Vector DNA
Serves as the carrier of the DNA segment that is to be cloned
Can replicate independently of the host chromosomal DNA
To prepare chromosomal DNA, the scientist has to
Obtain cellular tissue from the organism of interest
Break open the cells
Extract and purify DNA using a variety of biochemical techniques
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4. The cell that harbors the vector is called the host cell
When a vector is replicated inside a host cell, the DNA that it carries is also replicated
The sequence of the origin of replication determines whether that vector can replicate in a particular host cell
The vectors commonly used in gene cloning were originally derived from two natural sources
1. Plasmids
2. Viruses
Many naturally occurring plasmids have selectable markers
Typically, genes conferring antibiotic resistance to the host cell
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5. Cloning Experiments Involve Enzymes that Cut and Paste DNA
Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments
The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes
These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand
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6. Restriction enzymes bind to specific DNA sequences
These are typically palindromic
The sequence is identical when read in the opposite direction in the complementary strand
For example, the EcoRI recognition sequence is
5’ GAATTC 3’
3’ CTTAAG 5’
Some restriction enzymes digest DNA into fragments with “sticky ends” These DNA fragments will hydrogen bond to each other due to their complementary sequences
Other restriction enzymes generate blunt ends
The enzyme NaeI
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7. Cleavage by restriction enzymes is the first step to making recombinant DNA. In this case, the ends are ‘sticky’ in that they have a short, single-stranded end that can base-pair with another piece of DNA cut with the same enzyme.

10. Restriction enzymes are made naturally by many species of bacteria
Restriction enzymes were discovered in the 1960s and 1970s by Werner Arber, Hamilton Smith and Daniel Nathans
Restriction enzymes are made naturally by many species of bacteria
They protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophage
Currently, several hundred different restriction enzymes are available commercially
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11. A recombinant DNA molecule
This interaction is not stable because it involves only a few hydrogen bonds
To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked
A recombinant DNA molecule

13. The Steps in Gene Cloning
The procedure shown seeks to clone the human b-globin gene into a plasmid vector
The vector carries two important genes
ampR  Confers antibiotic resistance to the host cell
Identifies cells that have taken up the vector
lacZ  Encodes b-galactosidase
Provides a means by which bacteria that have picked up the cloned gene can be identified
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14. This is termed a hybrid vector
Digestion of a human cell would actually produce tens of thousands of fragments.
This is termed a hybrid vector

15. Cells that are able to take up DNA are called competent cells
This step of the procedure is termed transformation, when plasmid vectors are used, and transfection, when a viral vector is introduced into a host cell
Cells that are able to take up DNA are called competent cells
X-Gal is an analog of lactose
IPTG – molecular mimic of allolactose
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17. All bacterial colonies growing on the plate had to have picked up the vector and its ampR gene
But how to differentiate between the colonies that have a circularized vector from those with a hybrid vector?
In the hybrid vector, the chromosomal DNA inserts into the lacZ gene, thereby disrupting it
By comparison, the recircularized vector has a functional lacZ gene
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18. The growth media contains two relevant compounds:
IPTG (isopropyl-b-D-thiogalactopyranoside)
A lactose analogue that can induce the lacZ gene
X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside)
A colorless compound that is cleaved by b-galactosidase into a blue dye
The color of bacterial colonies will therefore depend on whether or not the b-galactosidase is functional
If it is, the colonies will be blue
If not, the colonies will be white
In this experiment
Bacterial colonies with recircularized vectors form blue colonies
While those with hybrid vectors form white colonies
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19. The net result of gene cloning is to produce an enormous amount of copies of a gene
During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector
Amplification of the gene occurs in two ways:
1. The vector gets replicated by the host cell many times
This will generate a lot of copies per cell (25-50 for plasmids)
2. The bacterial cell divides approximately every 20 minutes
This will generate a population of many million overnight
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20. cDNA To clone DNA, one can start with a sample of RNA
The enzyme reverse transcriptase is used
Uses RNA as a template to make a complementary strand of DNA
From retroviruses to copy their RNA genome to DNA
DNA that is made from RNA is called complementary DNA (cDNA)
It could be single- or double-stranded
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21. polyA tail

22. This has two ramifications
From a research perspective, an important advantage of cDNA is that it lacks introns
This has two ramifications
1. It allows researchers to focus their attention on the coding sequence of a gene
2. It allows the expression of the encoded protein
Especially, in cells that would not splice out the introns properly
(e.g., a bacterial cell)
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23. Polymerase Chain Reaction
Another way to copy DNA is a technique called polymerase chain reaction (PCR)
It was developed by Kary Mullis in 1985
Unlike gene cloning, PCR can copy DNA without the aid of vectors and host cells
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24. Chromosomal DNA
Gene of interest
Primer binding
near one end
of the gene
A different primer
binding near the other
end of the gene
Many PCR cycles
Many copies
of the gene of
interest, flanked
by the regions
where the
primers bind.
(a) The outcome of a PCR experiment
Template
DNA
Site where reverse primer binds 5′ 3′ 3′ 5′
Site where forward primer binds
Denaturation: Separate DNA
strands with high temperature. 5′ 3′ 3′ 5′
Primer annealing: Lower
temperature, which allows primers
to bind to template DNA. 5′ 3′
Forward primer 3′ 5′ 5′ 3′
Reverse primer 5′ 3′ 3′ 5′ T G C A C C A G C A T C C T C A C G T G G T C G T A G G G A C T A G
Primer extension: Incubate at a
temperature that allows DNA
synthesis to occur. 3′ 5′
Reverse primer 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′
(b) The 3 steps of a PCR cycle
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25. The starting material for PCR includes
1. Template DNA
Contains the region that needs to be amplified
2. Oligonucleotide primers
Complementary to sequences at the ends of the DNA fragment to be amplified
These are synthetic and about nucleotides long
3. Deoxynucleoside triphosphates (dNTPs)
Provide the precursors for DNA synthesis
4. Taq polymerase
DNA polymerase isolated from the bacterium Thermus aquaticus
This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases
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27. Binding of the primers to the DNA is called annealing
PCR is carried out in a thermocycler, which automates the timing of each cycle
All the ingredients are placed in one tube
The experimenter sets the machine to operate within a defined temperature range and number of cycles
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28. A typical PCR run is likely to involve 20 to 30 cycles of replication
The sequential process of denaturing-annealing-synthesis is then repeated for many cycles
A typical PCR run is likely to involve 20 to 30 cycles of replication
This takes a few hours to complete
After 20 cycles, a DNA sample will increase 220-fold (~ 1 million-fold)
After 30 cycles, a DNA sample will increase 230-fold (~ 1 billion-fold)
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30. RT-PCR is carried out in the following manner
PCR is also used to detect and quantitate the amount of RNA in living cells
The method is called reverse transcriptase PCR (RT-PCR)
RT-PCR is carried out in the following manner
RNA is isolated from a sample
It is mixed with reverse transcriptase and a primer that will anneal to the 3’ end of the RNA of interest
This generates a single-stranded cDNA which can be used as template DNA in conventional PCR
RT-PCR is extraordinarily sensitive
It can detect the expression of small amounts of RNA in a single cell
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31. 3′ 3′ 5′
RNA isolated
from a sample
of cells 3′ 5′ 3′
5’ ′ 5′ 5′ 3′
RNA of interest
Add reverse transcriptase, a primer
that binds near the 3′ of the RNA of
interest, and deoxyribonucleotides 3′ 3′ 5′ 3′ 5′ 3′ 5′ 5′ 3′ 5′ 3′ 5′
Subject to PCR as described
in Figures 18.5 and 18.6
Primer
Double-stranded
cDNAs derived
from the RNA
of interest
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32. DNA LIBRARIES AND BLOTTING METHODS
Molecular geneticists usually want to study particular genes within the chromosomes of living species
This presents a problem, because chromosomal DNA contains thousands of different genes
The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes
Scientists have also developed techniques to identify gene products
RNA that is transcribed from a particular gene
Protein that is encoded in an mRNA
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33. DNA Libraries
A DNA library is a collection of thousands of different cloned fragments of DNA
When the starting material is chromosomal DNA, the library is called a genomic library
A cDNA library contains hybrid vectors with cDNA inserts
Should represent the genes expressed in the cells the RNA was isolated from
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35. Copyright ©The McGraw-Hill Companies, Inc
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36. In most cloning experiments, the ultimate goal is to clone a specific gene
For example, suppose that a geneticist wishes to clone the rat b-globin gene
Only a small percentage of the hybrid vectors in a DNA library would actually contain the gene
Therefore, geneticists must have a way to distinguish those rare colonies from all the others
This can be accomplished by using a DNA probe in a procedure called colony hybridization
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38. But how does one obtain the probe?
If the gene of interest has been already cloned, a piece of it can be used as the probe
If not, one strategy is to use a probe that likely has a sequence similar to the gene of interest
For example, use the rat b-globin gene to probe for the b-globin gene from another rodent
What if a scientist is looking for a novel type of gene that no one else has ever cloned from any species?
If the protein of interest has been previously isolated, amino acid sequences are obtained from it
The researcher can use these amino sequences to design short DNA probes that can bind to the protein’s coding sequence
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40. Stained DNA gel under UV

42. Southern Blotting
Southern blotting can detect the presence of a particular gene sequence within a mixture of many
It was developed by E. M. Southern in 1975
Southern blotting has several uses
1. It can determine copy number of a gene in a genome
2. It can detect small gene deletions that cannot be detected by light microscopy
3. It can identify gene families
4. It can identify homologous genes among different species
5. It can determine if a transgenic organism is carrying a new or modified gene
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43. Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene, has been cloned
This cloned DNA is labeled (e.g., radiolabeled) and used as a probe
The probe will be able to detect the gene of interest within a mixture of many DNA fragments
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44. Gel electrophoresis of total DNA digested with restriction enzyme and subjected to Southern blot hybridization

45. Northern Blotting
Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules
It was not named after anyone called Northern!
Originally known as ‘Reverse-Southern’ which became Northern.
Northern blotting has several uses
1. It can determine if a specific gene is transcribed in a particular cell type
Nerve vs. muscle cells
2. It can determine if a specific gene is transcribed at a particular stage of development
Fetal vs. adult cells
3. It can reveal if a pre-mRNA is alternatively spliced
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46. Northern blotting is rather similar to Southern blotting
It is carried out in the following manner
RNA is extracted from the cell(s) and purified
It is separated by gel electrophoresis
It is then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a radioactive probe
The filters are then exposed to an X-ray film
RNAs that are complementary to the radiolabeled probe are detected as dark bands on the X-ray film
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47. The three mRNAs have different molecular weights
Smooth and striated muscles produce a larger amount of tropomyosin mRNA than do brain cells
This is expected because tropomyosin plays a role in muscle contraction
The three mRNAs have different molecular weights
This indicates that the pre-mRNA is alternatively spliced
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48. Western Blotting
Western blotting is used to identify a specific protein within a mixture of many protein molecules
Again, it was not named after anyone called Western!
Western blotting has several uses
1. It can determine if a specific protein is made in a particular cell type
Red blood cells vs. brain cells
2. It can determine if a specific protein is made at a particular stage of development
Fetal vs. adult cells
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49. Western blotting is carried out as follows:
Proteins are extracted from the cell(s) and purified
They are then separated by SDS-PAGE
They are first dissolved in the detergent sodium dodecyl sulfate
This denatures proteins and coats them with negative charges
The negatively charged proteins are then separated by polyacrylamide gel electrophoresis
They are then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a primary antibody (recognizes the protein of interest)
A secondary antibody, which recognizes the constant region of the primary antibody, is then added
The secondary antibody is also conjugated to alkaline phosphatase
The colorless dye XP is added
Alkaline phosphatase converts the dye to a black compound
Thus proteins of interest are indicated by dark bands

50. Western blot analysis

51. Western blot analysis

52. This experiment indicates that b-globin is made in red blood cells but not in brain or intestinal cells
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53. Techniques that Detect the Binding of Proteins to DNA or RNA
Researchers often want to study the binding of proteins to specific sites on a DNA or RNA molecule
For example, the binding to DNA of transcription factors
To study protein-DNA interactions, the following two methods are used
1. Gel retardation assay
Also termed band shift assay
2. DNA footprinting
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54. The technical basis for a gel retardation assay is this:
The binding of a protein to a fragment of DNA retards its rate of movement through a gel
Higher mass and therefore slow migration
Lower mass and therefore fast migration
Gel retardation assays must be performed under nondenaturing conditions
Buffer and gel should not cause the unfolding of the proteins nor the separation of the double helix
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55. DNA Sequencing
During the 1970s two DNA sequencing methods were devised
One method, developed by Alan Maxam and Walter Gilbert, involves the base-specific cleavage of DNA
The other method, developed by Frederick Sanger, is known as dideoxy sequencing
The dideoxy method has become the more popular and will therefore be discussed here
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57. The dideoxy method is based on our knowledge of DNA replication but uses a clever twist
DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other. Nucleotides missing that 3’–OH can be synthesized
Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow
This is referred to as chain termination
If ddATP is used, termination will always be at an A in the DNA
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58. This is accomplished using cloning or PCR techniques
Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts
This is accomplished using cloning or PCR techniques
In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site
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59. Copyright © The McGraw-Hill Companies, Inc
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A G G C T T C C
Sequence to
be analyzed
(target DNA) G T T
Primer G A
The newly-made DNA fragments can be separated according to their length by running them on an acrylamide gel
Annealing
site C 5′
Recombinant vector
They can then be visualized as fluorescence peaks as the bands run off the bottom of the gel
Many copies of the recombinant vector, primer,
dNTPs, fluorescently labeled dideoxynucleotides,
and DNA polymerase are mixed together.
Incubate to allow the synthesis of DNA.
CACCGTAAGGACTddG
CACCGTAAGGACddT
CACCGTAAGGAddC
CACCGTAAGGddA
CACCGTAAGddG
CACCGTAAddG
CACCGTAddA
CACCGTddA
CACCGddT
CACCddG
CACddC
CAddC
CddA
ddC
Nucleotides added to primer
Separate newly made strands by
gel electrophoresis.
Sequence
deduced
from gel G T C A C A C C G T A A G G A C T G G G A A T G C C
Laser
beam A C
Fluorescence
detector
(a) Automated DNA sequencing
(b) Output from automated sequencing

60. An important innovation in the method of dideoxy sequencing is automated sequencing
It uses a single tube containing all four dideoxyribonucleotides
However, each type (ddA, ddT, ddG, and ddC) has a different-colored fluorescent label attached
After incubation and polymerization, the sample is loaded into a single lane of a gel
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61. The procedure is automated using a laser and fluorescent detector
The fragments are separated by gel electrophoresis
Indeed, the mixture of DNA fragments are electrophoresed off the end of the gel
As each band comes off the bottom of the gel, the fluorescent dye is excited by the laser
The fluorescence emission is recorded by the fluorescence detector
The detector reads the level of fluorescence at four wavelengths