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AGRO/ANSC/BIOL/GENE/HORT 305
Fall, 2016
Recombinant DNA Technology (Chpt 20, Genetics by Brooker)
Lecture outline: (#14)
- 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. 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.
GENE CLONING
- The term gene cloning refers to the phenomenon of isolating and making many copies
of a gene
Table 20.1 summarizes some of the more common uses of gene cloning
- 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.
- 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 vectors commonly used in gene cloning were originally derived from two natural
sources:
o 1. Plasmids
o 2. Viruses
- Commercially available plasmids have selectable markers, typically, genes
conferring antibiotic resistance to the host cell. Plasmids have an origin of
replication that is recognized in specific host cells.
- Viruses can infect living cells and propagate themselves by taking control of
the host’s metabolic machinery. When a gene is inserted into a viral genome,
the gene is replicated every time the viral DNA is replicated.
Table 20.2 provides a general description of several vectors used to clone small segments
of 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.
Figure 20.1 shows the action of a restriction endonuclease.
- Restriction enzymes were discovered in the 1960s and 1970s by Werner Arber,
Hamilton Smith and Daniel Nathans. (Nobel Prize in 1978)
- 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
Table 20.3 gives a few examples
- 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’
CTTAAG
- 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
For example, the enzyme NaeI (Refer to Table 20.3)
THE STEPS IN GENE CLONING:
The general strategy followed in a typical cloning experiment is outlined in Figure 20.2
- The vector carries two important genes.
o ampR  Confers antibiotic resistance to the host cell
o lacZ  Encodes -galactosidase
- Provides a means by which bacteria that have picked up the cloned gene can
be identified
- All bacterial colonies growing on the plate had to have picked up the vector
and its ampR gene
- 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.
- The growth media contains two relevant compounds:
o IPTG (isopropyl--D-thiogalactopyranoside): A lactose analogue that
can induce the lacZ gene.
o X-Gal (5-bromo-4-chloro-3-indoyl--D-galactoside): A colorless
compound that is cleaved by -galactosidase into a blue dye
- The color of bacterial colonies will, therefore, depend on whether or not the galactosidase is functional:
o If it is, the colonies will be blue
o 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
- 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.
2. The bacterial cell divides approximately every 30 minutes. This will generate a
population of many million overnight.
- Recombinant DNA technology is not only used to clone genes. Sequences such as
telomeres, centromeres and highly repetitive sequences can be cloned as well.
cDNA can be made from mRNA via Reverse Transcriptase
- 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
- DNA that is made from RNA is called complementary DNA (cDNA)
- It could be single- or double-stranded
Synthesis of cDNA is presented in Figure 20.3
- From a research perspective, an important advantage of cDNA is that it
lacks introns. This has two ramifications:
- It allows researchers to focus their attention on the coding sequence of a gene
- It allows the expression of the encoded protein. Especially, in cells that would
not splice out the introns properly (e.g., a bacterial cell).
RESTRICTION MAPPING:
- Sometimes, it is necessary to obtain smaller clones from a large chromosomal
DNA insert. This process is termed subcloning.
- Cloning and subcloning require knowledge of the locations of restriction
enzyme sites in vectors and hybrid vectors.
- A common approach to examine the locations of restriction sites is known as
restriction mapping.
Figure 18.4 outlines the restriction mapping of a bacterial plasmid, pBR322.
- The restriction map can be deduced by comparing the sizes of DNA fragments
obtained from the single, double and triple digestions.
- Another way to obtain a restriction map is via DNA sequencing. Computer
programs can scan the DNA sequence of the hybrid vector and identify
restriction enzyme sites.
POLYMERASE CHAIN REACTION:
- Another way to copy DNA is a technique called polymerase chain reaction
(PCR). It was developed by Kary Mullis in 1985. Was awarded the Nobel
prize in 1993.
- Unlike gene cloning, PCR can copy DNA without the aid of vectors and host
cells.
The PCR method is outlined in Figures 20.5, 20.6
- The starting material for PCR includes:
o Template DNA: should contain the region that needs to be amplified
o Oligonucleotide primers; Complementary to sequences at the ends of
the DNA fragment to be amplified. These are synthetic and about 1520 nucleotides long.
o Deoxynucleoside triphosphates (dNTPs). Provide the precursors for
DNA synthesis.
o 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
- 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.
- 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)
- For a PCR reaction, a researcher must have prior knowledge about the sequence of the
template DNA, in order, to construct the synthetic primers.
- 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: (Figure 20.7)
o RNA is isolated from a sample
o It is mixed with reverse transcriptase and a primer that will anneal to
the 3’ end of the RNA of interest.
o 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.
DETECTION OF GENES AND GENE PRODUCTS
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. There are several different techniques
to choose from.
DNA libraries
- A DNA library is a collection of thousands of cloned fragments of DNA.
o When the starting material is chromosomal DNA, the library is called
a genomic library.
o A cDNA library contains hybrid vectors with cDNA inserts
The construction of DNA libraries is shown in Figure 20.11
Screening of libraries:
- In most cloning experiments, the ultimate goal is to clone a specific gene.
For example, suppose that a geneticist wishes to clone the rat -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.
Refer to Figure 20.12
The DNA 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 -globin gene to probe for the -globin gene from
another rodent.
- 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. These amino sequences can be used to design short DNA probes that can bind to the
protein’s coding sequence.
Southern Blotting is used to detect DNA sequences
- 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
Prior to a Southern blotting experiment, the gene of interest, or a fragment of a gene,
should have 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
The technique of Southern Blotting is shown in Figure 20.15
- Northern blotting is used to identify a specific RNA within a mixture of many RNA
molecules
- 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
Figure 20.16 shows the results of a Northern blot for mRNA encoding a protein called
tropomyosin
- 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.
- Western blotting is used to identify a specific protein within a mixture of many protein
molecules
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
Western blotting is carried out as such:
- 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
Figure 20.17 shows the results of a Western blot for the -globin polypeptide
Techniques that Detect the Binding of Proteins to DNA
- To study protein-DNA interactions, the following two methods are used
o 1. Gel retardation assay. Also termed band shift assay.
o 2. DNA footprinting
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. (Figure 20.18)
- 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
The technical basis for DNA footprinting is this:
- A segment of DNA that is bound by a protein will be protected from digestion
by the enzyme DNase I
ANALYSIS & ALTERATION OF DNA SEQUENCES
- Analyzing and altering DNA sequences is a powerful approach to
understanding genetics
- A technique called DNA sequencing enables researchers to determine the
base sequence of DNA. It is one of the most important tools for exploring
genetics at the molecular level.
- Another technique known as site-directed mutagenesis allows scientists to
change the sequence of DNA. This too provides information regarding the
function of genes
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. (Figure 20.12)
- The dideoxy method is based on our knowledge of DNA replication with
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.
-
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
If double-stranded DNA is used as the template, it must be denatured at the
beginning of the experiment
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.
The procedure is automated using a laser and fluorescent detector.
The fragments are separated by gel electrophoresis, 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