Recombinant DNA Technology

Restriction enzymes

Isolated from various bacteria, restriction enzymes recognize short DNA sequences
and cut the DNA molecules at those specific sites. (A natural biological function of
these enzymes is toprotect bacteria by attacking viral and other foreign DNA.) Cuts
yield either "staggered" or "sticky" ends (see figure) or "blunt" ends. Two pieces of
DNA cut with the same enzyme, can be pasted together using another enzyme called
"DNA ligase".

Some restriction enzymes cut

the DNA very infrequently
(rare-cutters), generating a
small number of very large
fragments (several thousand to a
million base pairs). Most
enzymes cut DNA more
frequently, thus generating a
large number of small fragments
(less than a hundred to more
than a thousand bp).

On average, restriction enzymes

with

* 4- base recognition sites will

yield pieces 256 bases long,

* 6- base recognition sites will

yield pieces 4000 bases long,
and

* 8- base recognition sites will yield pieces 64,000 bases long.

Why?

Since hundreds of different restriction enzymes have been characterized, DNA can
be cut into many different small fragments.

Agarose gel electrophoresis

DNA fragments that have been cut with REs can be separated based on their relative
sizes by running them in a gel made of the polymer agarose, a jelly-like substance.
DNA, which overall has a negative charge, is loaded into a well and current that
runs from the negative to the positive poles is applied. DNA bands will separate
with the smaller fragments running ahead of the bigger ones. A standard with DNA
pieces of known sizes is run in parallel to estimate size of experimental DNAs.
DNA is visualized by staining the gel with fluorescent ethidium bromide (EtBr).
EtBr is hydrophobic substance that binds to the bases in double helix. Size can be
more precisely established by interpolating the distance migrated by the
experimental DNA into a plot charting size (in base-pairs) of the known standards
in a semi-log scale on the Y-axis vs their migrated distance in the gel (in mm or cm
or inches) in the X-axis. Resolution depends on the concentration of agarose used
(usually 1%), the length of the gel and on the pattern of current applied.

DNA sequencing

Two methods: the chemical method (Maxam and Gilbert) and the enzymatic or
chain termination method (Sanger). The latter has become the standard procedure
for sequencing DNA. This method is rapid, reliable, easy and accurate allowing the
indirect determination of a proteinís amino acid sequence by determining the
nucleotide sequence of its coding DNA gene. Automation of this method has made
possible the various genome sequencing projects, from small viruses, to E.coli to
yeast and ultimately, it will allow the whole sequencing of the ~3 x 109 bps in the
human genome. Explain method (Figure 7-8).

Hybridization techniques

The principle of complementary base pairing introduced by Watson and Crick (A-T;
G-C) is the basis of a series of techniques developed to detect, among a mixture of
nuclei acids (DNA or RNA) immobilized onto a solid support , a specific target
fragment or sequence. The target, in the appropiate conditions, will base-pair
according to W/C rules with another piece of Nucleic Acid (the probe) that has its
complementary nucleotide sequence. The probe is labelled for example with
radioactive nucleotides, so that, after the proper washing out of unspecific products,
the target can be detected by its binding to the labelled probe (using for example,
autoradiography).
Depending on the nature of the target, hybridizations techniques include:

Southern bloting- DNA target/DNA probe-invented by the English Molecular

Biologist E.M. Southern

Northern bloting- RNA target/DNA probe

Western blot-Protein target/antibody protein probe. The interaction between probe

and target in a Western blot does not involve W/C base paring. Here, the
biochemical complementary is between two protein surfaces and may involve non-
covalent associations such as H-bonding or Ionic interactions.

SouthWestern blot-DNA target/Protein probe.

The following table summarize the major features of each hybridization technique.

In situ hybridization- either DNA or RNA probes are used to locate specific nuclei
acid sequences in the cell, i.e. genes on chromosomes or mRNA if whatever
compartment. Information about the temporal/spatial distribution of a mRNA can
provide valuable insights about the function of the gene that encodes it. To increase
spatial resolution non-radioactive methods of labelling the probes are preferred
nowadays. However, some of these yield lower sensitivity than the radioactive
probes.

Genetic maps

A genetic linkage map shows the RELATIVE locations of specific DNA markers
along the chromosome. Any inherited physical or molecular characteristic that
differs among individuals and is easily detectable in the laboratory is a potential
genetic marker. Markers can be expressed DNA regions (genes) or DNA segments
that have no known coding function but whose inheritance pattern can be followed.
DNA sequence differences are especially useful markers because they are plentiful
and easy to characterize precisely.

Markers must be
polymorphic to be useful in
mapping; that is, alternative
forms must exist among
individuals so that they are
detectable among different
 members in family studies.
Polymorphisms are
variations in DNA sequence
(mutations)that occur on
average once every 300 to
500 bp. Variations within
coding sequences can lead
to observable changes, such
as differences in eye color,
blood type, and disease
susceptibility. Most variations occur within non-coding regions and have little or no
effect on an organisms appearance or function, yet they are detectable at the DNA
level and can be used as markers. Examples of these types of markers include
(1)restriction fragment length polymorphisms (RFLPs), which reflect sequence
variations in DNA sites that can be cleaved by DNA restriction enzymes (see
below), and (2)variable number of tandem repeat sequences, which are short
repeated sequences that vary in the number of repeated units and, therefore, in
length (a characteristic easily measured). The human genetic linkage map is
constructed by observing how frequently two markers are inherited together which
is determined by the recombination rate of the particular region. The closer the
markers are to each other the more tightly-linked the less likely a recombination
event will fall between and separate them. Recombination frequency thus provides
an estimate of the distance between two markers.

On the genetic map, distances between markers are measured in terms of

centimorgans (cM), named after the American geneticist Thomas Hunt Morgan.
Two markers are said to be 1cM apart if they are separated by recombination 1% of
the time. A genetic distance of 1cM is roughly equal to a physical distance of 1
million bp (1 Mb). Remember recombination frequency is not constant across the
genome. It varies between regions of chromosomes. (Include Drosophila example,
Genes V).

The value of the genetic map is that an inherited disease can be located on the map
by following the inheritance of a DNA marker present in affected individuals (but
absent in unaffected individuals), even though the molecular basis of the disease
may not yet be understood nor the responsible gene identified. Genetic maps have
been used to find the chromosomal location of several important disease genes,
including cystic fibrosis, sickle cell disease, Tay-Sachs disease, fragile X syndrome,
and myotonic dystrophy.

(How disease genes are found.. Insert Fig 6.10 Genes V).

Physical Maps

Physical maps vary in their degree of resolution. The lowest-resolution physical

map is the chromosomal (cytogenetic) map, which is based on the distinctive
banding patterns observed by light microscopy of stained chromosomes. A cDNA
map shows the locations of expressed DNA regions (exons) on the chromosomal
map. The more detailed cosmid contig map depicts the order of overlapping DNA
fragments spanning the genome. A macrorestriction map describes the order and
distance between enzyme cutting (cleavage) sites. The highest-resolution physical
map is the complete elucidation of the DNA base-pair sequence of each
chromosome in the human genome.

Vectors, DNA cloning and libraries.

A clone describes a large number of identical cells or molecules with a single

ancestral cell or molecule. Cloning vectors were developed from bacterial
endogenous extrachromosomal or virus elements to shuttle or carry an inserted piece
of DNA or a gene, for the purposes of producing more of it or of its protein product
inside a bacterial host. RE are used to insert the foreign DNA into the vector. Types
of vectors: plasmid, cosmids, yeast artificial chromosomes(YACs). Each vector was
design to carry larger chunks of DNA with plasmids about 10 Kbp, Cosmids about
40Kbp and YAC about 1000Kbp, maximum. The example of the figure illustrates
the cloning of one of the first human genes: the insulin gene.

Cloning a Gene by Hybridization:

In this case, 'to clone the actin gene from humans' means "to end up with a plasmid
which contains a fragment of human DNA which includes the actin gene". The
usual starting point is a plasmid clone of the actin gene from another organism and
human chromosomal DNA. DNA-DNA hybridization is usually used for this.

Although Southern blotting involves DNA-DNA hybridization, it is not a useful

procedure for cloning a gene. If we were to cut human DNA with a restriction
enzyme and run it on a Southern blot probed with a clone of the actin gene from an
other organism, we could construct a restriction map of the human actin gene.
However, we can not isolate the human actin gene DNA from either the gel or the
filter because at each molecular weight on the gel, there are many bands of the same
length but different sequences.

For this reason, separating the DNA fragments by molecular weight is unsuitable.
Instead, we separate them by sequence - by making a LIBRARY, we end up with a
collection of plasmids, physically separated, each containing a different fragment of
human DNA. Here is a typical procedure: cloning the human gene for actin, given a
clone of the yeast actin gene.

1) Isolate genomic (chromosomal) DNA from human cells.

2) Create a plasmid library of human DNA restriction fragments (genomic library).

This results in a collection of bacterial colonies, each containing a different plasmid
with a different inserted piece of human DNA.

3) Plate the colonies on agar plates and let them grow. These are called the master
plates.

4) Press a piece of nitrocellulose onto each master plate and lift off. This leaves
some of each colony on the plate and a replica on the filter.

5) Break open (lyse) the bacteria on the filter under conditions that make their
plasmid DNA single-stranded, and bind the DNA onto the filter. There are now
spots of single-stranded plasmid DNA on the filter. These spots correspond to the
locations of the colonies of bacteria on the master plate.

6-9) "Screen" library by Southern hybridization , using a labeled restriction

fragment of the yeast actin gene from the plasmid as a probe. In all likelihood, the
stringency of the hybridization conditions would have to be "lowered" to allow
stable annealing between the yeast and human actin sequences which, although
similar, are surely not identical. The result will be a piece of X-ray film with a dark
spot corresponding to a place on the filter where DNA from the human actin gene
was present. This spot on the filter corresponds to a colony from the master plate.

10) Pick up some of the bacteria from the appropriate colony, grow them in broth
and extract their plasmid DNA. It contains a fragment of human DNA containing
the actin gene.

It is also possible to clone gene X using an antibody against the protein produced by
gene X. In this case, you make an EXPRESSION LIBRARY - a library where the
vector contains a strong E. coli promoter and an ATG codon. Cells containing these
plasmids will produce large amounts of protein from whichever human gene they
carry. If you prepare the filter replicas of the library as above, you can probe them
with an antibody to find the colony that contains a plasmid that expresses protein X.
This plasmid will contain gene X.

An expression library is a kind of cDNA Library. The starting material is the mRNA
from a given tissue or cell. DNA copies of the mRNA(cDNA) are made using
Reverse Transcriptase (RT), the same enzyme that allows retroviruses to replicate
their RNA genome. The cDNA is the clone into a particular vector to generate the
library. Subtractive hybridization is a technique to enrich for particular mRNA
sequences prior to cDNA cloning. The goal is to eliminate sequences present in two
samples and to be left with the sequences that are unique to a particular sample (i.e.,
cultured cells treated with a drug vs untreated cells. Most mRNAs, except those for
the genes induced/repressed by the drug, will be present in these two populations.
You would like to get rid of the former and enrich for the latter).

DNA Amplification: PCR technology

Described as being to genes what Gutenberg's printing press was to the written
word, PCR (or Polymerase chain reaction can amplify a desired DNA sequence of
any origin (virus, bacteria, plant, or human) hundreds of millions of times in a
matter of hours, a task that would have required several days with recombinant
DNA technology. PCR is especially valuable because the reaction is highly specific,
easily automated, and capable of amplifying minute amounts of sample. For these
reasons, PCR has also had a major impact on clinical medicine, genetic disease
diagnostics, forensic science, and evolutionary biology.

PCR is a process based on a heat-resistant DNA polymerase enzyme (purified from

bacteria that lives in hot springs), which can synthesize a complementary strand to a
given DNA strand in a mixture containing the 4 DNA bases and 2 DNA fragments
(primers, each about 20 bases long) flanking the target sequence. The mixture is
heated to separate the strands of double-stranded DNA containing the target
sequence and then cooled to allow (1) the primers to find and bind to their
complementary sequences on the separated strands and (2) the polymerase to extend
the primers into new complementary strands. Repeated heating and cooling cycles
multiply the target DNA exponentially , since each new double strand separates to
become two templates for further synthesis. In about 1 hour, 20 PCR cycles can
amplify the target by a million-fold.
DNA Engineering.
Heterologuos expression. Once a cDNA (partial- or full-length) for a
gene has been cloned, the analysis of functional properties of the
protein it codes for demands producing it in relatively high quantities.
Proteins can be expressed in bacteria, yeast, xenopus oocytes, and most
often in (mammalian) cell lines that can be easily grown in the lab, and
to which DNA constructs can be readily introduced, either transiently
or permanently, by transfection. Special purpose vectors called
Expression vectors are used. The cDNA is cloned downstream a strong
promoter, (for mammalian hosts) usually derived from a virus (e.g.
CMV or RSV). The vector also provides poly A+ -addition signals and
other cis-acting signals necessary for replication and selection in either
bacteria or the cell line used. DNA introduced by transient transfection
is supercoiled DNA that replicates as an extrachromosomal element.
DNA introduced by permanent transfection is linear DNA that has to
integrate into the host genome. Cells that carry this construct have to be
selected for, usually by addition of an antibiotic (e.g. neomycin) to the
growth medium for which the expression vector carries a protein that
confers resistance (i.e. allows growth) to the cell. (Show pcDNA3).
Mutations in the cDNA can be introduced by reverse genetics
techniques and these can be expressed as above to reveal insights about
how the protein works
Transgenic animals. Cloned genes can also be expressed in vivo if
introduced into the germ line of an animal. Mice and fruit flyes are the
most used "host". Such an animal is called a "transgenic organism"
and the introduced engineered gene a "transgene". (Go through
method). Most of the time you want to examine what happens if you
"overexpress" the protein in the same tissue where it is usually
expressed or, also, in another tissue where it is not usually expressed.
Thus, a strong promoter from a gene known to be expressed in a given
tissue is chosen to drive transgene expression. Trangenics have
provided numerous clues about the function of proteins in vivo and are
used to develop experimental models for diseases like cancer.
Knock out mice. Just as you can overexpress a protein in vivo, you can
delete part or the whole of the endogenous gene that encodes it by gene
targeting inactivation. In this case, the technique makes use of
homologue recombination to replace the normal gene for the defective
one. You can then examine the functional consequences of not making
the protein in vivo (go through method). In many cases (for dominant
genes), lacking both copies of a gene is lethal and having just one
normal copy is as good as having both. Thus, knockouts are not very
informative. Hence, new more sophisticated techniques that allow
knocking out a gene either in a specific tissue or cell type or during a
particular developmental stage are now being used to overcome this
shortcoming (i.e.flox/cre recombination system). In any event, KO mice
are one of the methodologies that provide the most conclusive evidence
for the requirement of a particular gene/protein in a given process, and
is being used more and more.
Repoter gene constructs. When trying to determine what are the cis-
acting regulatory regions that control the expression (transcription)
levels of a gene, is usually routine to clone a large piece of DNA
containing these sequences and hook it to a downstream sequence that
encodes an easy-to-measure protein called a reporter. This is a lot
more convenient and accurate than trying to estimate the absolute levels
of mRNA. In vivo transgene reporter expression driven by the promoter
of interest will reveal the temporal/spatial pattern of expression of the
gene, measured by an easier method than the alternative in situ
hybridization. The most used reporters are: bacterial b-galactosidase,
chloramfenicol acetyl transferase (CAT), fire-fly luciferase and most recently, the green
fluorescent protein (GFP)which allows measurements in living cells or organisms. Part of
the whole of a protein rather than a promoter can also be fused in frame to this reporters
to find out where it localizes. Reporter gene must not being expressed endogenously in
the cell types that you are working on, so their background should be null.