Recombinant DNA refers to the creation of new combinations of DNA segments that are not found

together in nature. The isolation and manipulation of genes allows for more precise genetic analysis as
well as practical applications in medicine, agriculture, and industry.

Making recombinant DNA

Overview: Isolate DNA  Cut with restriction enzymes  Ligate into cloning vector  transform
recombinant DNA molecule into host cell  each transformed cell will divide many, many times to form
a colony of millions of cells, each of which carries the recombinant DNA molecule (DNA clone)

A. Isolating DNA
1. Crude isolation of donor (foreign) DNA is accomplished by isolating cells  disrupting lipid
membranes with detergents  destroying proteins with phenol or proteases  degrading RNAs with
RNase  leaving DNA at the end
2. Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or by boiling
cells which removes bacterial chromosomal DNA from plasmid DNA.
3. To get purer DNA from either (1) or (2), crude DNA is
a) Fractionated on a CsCl2 gradient
b) Precipitated with ethanol
c) Poured over a resin column that specifically binds DNA

B. Cutting DNA
1. DNA can be cut into large fragments by mechanical shearing.
2. Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are
endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain
at those points. A variety of RE have been isolated and are commercially available. Most cut at
specific palindromic sites in the DNA (sequence that is the same on both antiparallel DNA strands).
These cuts can be a staggered which generate “sticky or overhanging ends” or a blunt which generate
flush ends.

C. Joining DNA
Once you have isolated and cut the donor and vector DNAs, they must be joined together.
The DNAs are mixed together in a tube. If both have been cut with the same RE, the ends will match up
because they are sticky. DNA ligase is the glue of molecular genetics that holds the ends of the DNAs
together. DNA ligase creates a phosophodiester bond between two DNA ends.

D. Amplifying the recombinant DNA

To recover large amounts of the recombinant DNA molecule, it must be amplified. This is accomplished
by transforming the recombinant DNA into a bacterial host strain. (The cells are treated with CaCl2 
DNA is added  Cells are heat shocked at 42 C  DNA goes into cell by a somewhat unknown
mechanism.) Once in a cell, the recombinant DNA will be replicated. When the cell divides, the
replicated recombinant molecules go to both daughter cells which themselves will divide later. Thus,
the DNA is amplified.

DNA clone = A section of DNA that has been inserted into a vector molecule and then replicated in a
host cell to form many copies.

E. Vectors
1. Requirements for a cloning vector
a) Should be capable of replicating in host cell
b) Should have convenient RE sites for inserting DNA of interest
c) Should have a selectable marker to indicate which host cells received recombinant DNA molecule
d) Should be small and easy to isolate

2. Bacterial plasmids are small, circular DNA molecules that are separate from the rest of the
chromosome. They replicate independently of the bacterial chromosome. Useful for cloning DNA
inserts less that 20 kb (kilobase pairs). Inserts larger than 20 kb are lost easily in the bacterial cell.

3. Bacteriophage lambda (45 kb) contains a central region of 15 kb that is not required for replication or
formation of progeny phage in E. coli. Thus, lambda can be used as a cloning vector by replacing the
central 15 kb with 10-15 kb of foreign DNA. This is done as follows: mix RE cut donor DNA and lambda
DNA in test tube  ligate  use in vitro packaging mix that will assemble progeny phage carrying the
foreign DNA  infect E. coli with the phage to amplify
4. Cosmids are hybrids of phages and plasmids that can carry DNA fragments up to 45 kb. They can
replicate like plasmids but can be packaged like phage lambda.

5. Expression vectors are vectors that carry host signals that facilitate the transcription and translation of
an inserted gene. They are very useful for expressing eukaryotic genes in bacteria.

6. Yeast artificial chromosomes (YACS) are yeast vectors that have been engineered to contain a
centromere, telomere, origin of replication, and a selectable marker. They can carry up to 1,000 kb of
DNA. Since they are maintained in yeast (a eukaryote), they are useful for cloning eukaryotic genes
that contain introns. Also, eukaryotic genes are more easily expressed in a eukaryotic host such as
yeast.

7. Bacterial artificial chromosomes (BACS) are bacterial plasmids derived from the F plasmid. They are
capable of carrying up to 300 kb of DNA.
Timeline of important events happened in molecular biology and bioinformatics

From Darwin's "On the Origin of Species" till Morgan's "Theory of the Gene"

1859 Charles Darwin published the "On the Origin of Species", introducing that genetic evolution
allowed adaptation over time to produce organisms best suited to the environment

1865 Gregor Mendel investigated "traits" passed from parents to prodigy and coined the terms
dominant and recessive traits

1869 Johann Meisher isolated DNA from the nuclei of white blood cells

1875 Charles Darwin introduced "gemmules" as mechanism of inheritance

1902 Walter Sutton created term "gene" to describe "factors" located on chromosomes: he observed
chromosomal movement during meiosis and developed the chromosomal theory of heredity

1905-1908 William Bateson and Reginal Crudell Punnett demonstrated actions of some genes modify
action of other genes: the first time gene regulation was demonstrated

1910 Thomas Hunt Morgan was the first to recognise genes are carried on chromosomes: the basis for
modern genetics. He demonstrated the existence of sex-linked genes and expanded trait
linkage using "crossing-over"

1911 Alfred Sturtevant, mapped the locations of several fruit fly genes. This was the first genetic map

1926 Thomas Hunt Morgan published the "theory of the gene" based on Mendelian genetics

From the invention of electrophoresis till the cracking of the genetic code

1933 A new technique, electrophoresis, was introduced by Arne Tiselius for separating proteins in
solution

1937 Frederick Charles Bawden discovered tobacco mosaic virus RNA

1944 Barbara McClintock reported transposable elements: "jumping genes"

1946 Edward Tatum and Joshua Lederberg discovered that bacteria can exchange genetic material
directly through conjugation

Max Delbruck and Alfred Day Hershey discovered a combination of genetic material from
viruses: genetic recombination

1950 Erwin Chargaff found that amounts of adenine and thymine and cytosine and guanine in DNA
are always about the same. This is now called "Chargaff's Rules"

1952 Rosalind Franklin and Maurice Wilkins performed X-ray crystallography studies of DNA, providing
crucial information that led to the elucidation of the structure of DNA

1953 James Watson and Francis Crick proposed the double-stranded, helical, complementary, anti-
parallel model for DNA

1955 Frederick Sanger announced the first complete sequence of a protein, bovine insulin
Arthur Kornberg discovered and isolated DNA polymerase from E. coli bacteria

1956 Francis Crick and George Gamov worked out the "Central Dogma" to explain protein synthesis
from DNA: the DNA sequence codes for amino acid sequences and genetic information flows in
one direction - from DNA to mRNA to protein

1959 Francois Jacob and Jacques Monod discovered an important mechanism behind genetic
regulation: mappable control functions located on chromosomes in DNA sequence - named
"repressor" and "operon"
1961 Marshall Nirenberg, Heinrich Mathaei and Severo Ochoa cracked the "Genetic Code": a
sequence of three nucleotide bases (codon) determine each of amino acids

From the first Arabidopsis newsletter till the introduction of sequence similarity sequencing

1964 First Arabidopsis newsletter, the Arabidopsis Information Service, was published

1967 Mary Weiss and Howard Green found a technique for combining human cells and mouse cells
grown in one culture: somatic cell hybridization

The first evolutionary trees from protein sequences were set op by WM Fitch and E Margoliash

1970 Howard Temin and David Baltimore independently isolated reverse transcriptase, an enzyme that
can make DNA from RNA

Torbojorn Caspersson and Lore Zech discovered a method for staining mammalian
chromosomes to reveal banding

1972 Paul Berg used a restriction enzyme to cut DNA and ligase to past two DNA strands together to
form hybrid circular molecule. This was the first recombinant DNA molecule
First successful DNA cloning experiments

1973 Stanley Cohen and Herbert Boyer first successfully transfered DNA from one life form into another:
a spliced viral DNA and bacterial DNA to create a plasmid with dual antibiotic resistance

1974 Allan Maxam and Walter Gilbert (Harvard) and Frederick Sanger (U.K. Medical Research Council)
independently developed different methods for sequencing DNA

1975 Georges Kohler and Cesar Milstein fused antibody-producing B lymphocyte cells with tumor cells
that are "immortal" to produce the first monoclonal antibodies

Edwin Southern published the experimental details for the Southern Blot technique to identify
DNA fragments

1977 Bacteriophage FX-174 (5368 bp) was the first complete genome (DNA) to be sequenced

Richard Roberts’ and Phil Sharp’s labs showed that eukaryotic genes contain many interruptions,
called introns.

1978 Genentech successfully produced human insulin using recombinant DNA technology in E. coli

David Botstein discovered the use of restriction enzymes produces different fragments from one
person to another, RFLP: restriction fragment length polymorphisms

1980 Kary Mullis invented the polymerase chain reaction (PCR), a method for multiplying DNA
sequences in vitro

1981 Gordon and Ruddle (Ohio University) made the first transgenic mice by inserting genes from other
animals with DNA microinjection

Human mitochondral DNA sequenced (16569 bp)

1982 Phage lambda genome sequenced (48,502 bp)

1983 First genetic modifed plant is created; a tobacco plant resistant to an antibiotic

1984 Alec Jeffreys developed the technique of using sequences of DNA for identification, called
"genetic fingerprinting"

Chiron Corp determined the entire sequence of the HIV-1 genome

1985 FASTP/FASTN introduced: algorithms sequence similarity searching

From the DNA fluorescence sequencer till the presentation of the human genome

1986 Applied Biosystems introduced the first automated DNA fluorescence sequencer

The Environmental Protection Agency (USA) approved the release of the first genetically
engineered crop: a herbicide resistant tobacco plants

1987 "Yeast artificial chromosomes", YACs, expression vector for large DNA fragments, were introduced
by David Burke

1988 National Centre for Biotechnology Information (NCBI) founded at NIH/NLM

EMBnet network for database distribution introduced

Clustal multiple alignment algorithm (Higgins) introduced

1990 Human Genome Project launched: estimated cost of $13 billion (plan 15 years)

BLAST: fast sequence similarity searching tool introduced by S. Karlin and S.F. Altshul

1991 EST: expressed sequence tag sequencing first described by Craig Venter and colleagues: a
method for identifying active genes

1992 The Institute for Genome Research (TIGR), associated with plans to exploit sequencing
commercially through gene identification and drug discovery, was formed

Mel Simon introduced the use of BACs for cloning

1993 The Sanger Centre, a genome research institute with the purpose to further the knowledge of
genomes, was crated in Hinxton, UK

The EMBL European Bioinformatics Institute, the centre for research and service for bioinformatics
was established in Hinxton, UK

1994 The FlavrSavr Tomato becomes the first genetic modified food to be approved for sale. A gene
expression the enzyme polygalacturonase, which is responsible for the tomato's softness, was
introduced by Calgene

1995 First completed sequence of a bacterial genome, Haemophilus influenzae by TIGR

1996 Yeast and E. coli genome completely sequenced

Arabidopsis Genome Initiative started

Patrick Brown of Stanford University presented the 'gene chip' containing 6116 different gene
specific sequences of the yeast genome

Ian Wilmut at Scotland's Roslin Institute presented"Dolly", a sheep cloned from the cell of an adult
mammary gland

1998 The genome of Caenerhabditis elegans, a small soil nematode, was completely sequenced
(97Mb)

1999 Drosophila melanogaster (fruitfly) genome completely sequenced (175 Mb)

2000 Completion of the Arabidopsis thaliana sequence (157 Mb)

Human genome draft version finished (3200 Mb)

2002 Presentation of human genome by Celera Genomics and the collaborating group of
laboratories supported by public foundation
Future goals of molecular biology and bioinformatics research

2010 Completion of the 2010 Project: the understanding the function of all genes within their cellular,
organismal and evolutionary context of Arabidopsis thaliana

2050 Completion of the first computational model of a complete cell, or maybe even already of a
complete organism