Recombinant DNA and

Biotechnology
gy
Sadava et al, 18. Recombinant DNA and
biotechnology

Molecular Cloning Toolbox - Restriction Enzymes

Restriction enzymes (restriction

endonucleases) cut double-stranded
DNA into smaller pieces.
Bacteria use these as defense against
DNA from bacteriophage and other
bacteria.
bacteria

Molecular Cloning Toolbox - Restriction Enzymes, Ligation

There are many restriction enzymes that cut DNA at

specific
p
base sequencesthe
q
recognition
g
sequence, or restriction site.
The EcoRI site (GAATTC), for example, is found at an
average of every 5kb in the human genome.
DNA fragments can be rejoined by DNA ligase.
Any two DNA sequences can be ligated together, thus
Recombinant DNA

Figure 18.2 Cutting, Splicing, and Joining DNA

[ligase uses ATP; done at 4 C]

Molecular Cloning - Inserting Genes into Cells

Recombinant DNA technology can be

used to clone,
clone or make exact copies of
genes.
The gene can be used to make a protein
((ie.,, translation)) but it must first be
inserted, (transfected [non-viral] or
]) into host cells.
transduced [[viral])
The altered host cell is called
t
transgenic.
i

Figure 18.3 Vectors for Carrying Recombinant DNA into Cells

Plasmids are often used

as cloning vectors
Plasmids are small
(~3 kb).
Genes for antibiotic
resistance can be
used to select for
bacteria that have
incorporated the
plasmid.
And they have an
origin of replication
and can replicate
p
independently.

Figure 18.11 Expression of Transgene in Host Cell Produces Large Amounts of its Protein Product

DNA cloning with

plasmid
Prepare recombinant
plasmid by cutting and
pasting
Insert into E.coli (or other
cell) and select using
antibiotic
Plasmid replicate within
cell and daughter cells
also contain plasmid
(freeze cells to save)
Isolate DNA by disrupting
cells and purify plasmid
and/or DNA fragment =
clones

Figure 18.5 Green Fluorescent Protein as a Reporter

Molecular Cloning Toolbox - Visualization of DNA

After DNA is cut, fragments of different sizes

can be separated by gel electrophoresis.
electrophoresis
Cleaved DNA is injected into a well of a porous
(agarose) gel
gel. An electric field is applied
across the gel. Negatively charged DNA
fragments move towards positive end.
Smaller fragments move faster than larger
ones.

Figure 15.8 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Figure 15.8 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

Molecular Cloning Toolbox - Visualization of DNA

Electrophoresis provides information on:

Size of fragments
fragments. Fragments of known
size (known as molecular weight
markers) provide comparison
comparison.
Presence of specific sequences. These
can be determined using probes
probes.
DNA is denatured while in the gel, then
transferred to a nylon filter to make a
blot.

Primers hybridize at T < melting

point
Primer (here labelled)
= Labelled probe

Tm of DNA depends on
composition
p
((GC p
pairs))

(annealing at 50-60 C)
http://www.discoveryandinnovation.com/BIOL202/notes/lecture22.html

Figure 15.16 Analyzing DNA Fragments by DNA Gel (Southern) Blotting

A probe has a known

sequence;
q
; hybridization
y
occurs at an appropriate
cool temperature
Southern blotting (also
Western blots for
proteins)
i )

Figure 18.6 Constructing Libraries

Libraries of all the

genes expressed in
a given cell type
can be made,
known as a cDNA
library
start with mRNA
(can also make
DNA libraries)
control plasmid to
E. coli ratio so that
one plasmid/cell;
each cell has only 1
fragment or 1
mRNA

Molecular Cloning - Making cDNA (coding DNA)

DNA libraries can be made from

complementary DNA (cDNA).
(cDNA)
mRNA is extracted from a tissue and the
poly A tails allowed to hybridize with
g dTa string
g of thymine
y
bases.
oligo
Oligo dT serves as a primer for reverse
t
transcriptase
i t
to
t synthesize
th i a
complementary DNA strand.

Synthesizing Complementary DNA

Primer + DNA polymerase

Molecular Cloning - cDNA Libraries

cDNA libraries are made from particular

ti
tissues
and
d representt a snapshot
h t off the
th
mRNA present at that time.
Used to compare gene expression in
different tissues at different stages of
development.
cDNA is also used to clone eukaryotic
genes.

Figure 18.9 DNA on a Chip

Microarray
(now use RNA-seq,
a PCR based
sequencing method)

Figure 18.10 Using DNA Arrays for Medical Diagnosis

Determine which genes (each spot

= different mRNA/cDNA) are
expressed more in tumour than in
normal (ie show up as red spots)
Some mRNAs are associated with
a good prognosis; these are markers
of good outcome

Summary
How is DNA replicated? [brief]

Replication of DNA is semiconservative. Each parent strand acts as a template for the synthesis of a new strand; thus the two
replicated DNA molecules each contain one parent strand and one newly synthesized strand.

In DNA replication, the enzyme DNA polymerase catalyzes the addition of nucleotides to the 3' end of each strand. Which
nucleotides are added is determined by complementary base pairing with the template strand.

Many proteins assist in DNA replication. DNA helicase separates the strands, and single-strand binding proteins keep the
strands from reassociating. Primase catalyzes the synthesis of a short RNA primer to which nucleotides are added by DNA
polymerase.

What are some applications of our knowledge of DNA structure and replication?

The polymerase chain reaction technique uses DNA polymerase to make multiple copies of DNA in the laboratory.

How are large DNA molecules analyzed?

Restriction enzymes, which are made by bacteria as a defense against viruses, bind to and cut DNA at specific recognition
sequences (also called restriction sites)
sites). These enzymes can be used to produce small fragments of DNA for study
study, a
technique known as restriction digestion

DNA fragments can be separated by size using gel electrophoresis.

Specific DNA sequences can be identified in a gel by probes with a complementary sequence in a procedure known as Southern
blotting.

What is recombinant DNA?

Recombinant DNA is formed by the combination of two DNA sequences from different sources.

Many restriction enzymes make staggered cuts in the two strands of DNA, creating fragments that have sticky ends with
unpaired bases.

DNA fragments
f
t with
ith sticky
ti k ends
d can be
b used
d to
t create
t recombinant
bi
t DNA if DNA molecules
l
l from
f
different
diff
t sources are cutt with
ith the
th
same restriction enzyme and spliced together with DNA ligase.

Summary
How are new genes inserted into cells?

One goal of recombinant DNA technology is to clone a particular gene, either for analysis or to produce its protein product in
q
quantity.
y

Bacteria, yeasts, and cultured plant cells are commonly used as hosts for recombinant DNA. Host cells into which
recombinant DNA is inserted, or transformed, are called transgenic cells.

To identify host cells that have taken up a foreign gene, the inserted sequence can be tagged with reporter genes, genetic
markers with easily identifiable phenotypes.

Vectors are DNA sequences that can carry new DNA into host cells
cells. Plasmids are one type of vector
vector.

What are the sources of DNA used in cloning?

DNA fragments from a genome can be inserted in host cells to create a gene library.

The mRNAs produced in a certain tissue at a certain time can be extracted and used to create complementary DNA (cDNA)
by reverse transcription.

What other tools are used to manipulate DNA?

DNA chip technology permits the screening of mRNA for thousands of sequences at the same time.

What is biotechnology?

Biotechnology is the use of living cells to produce materials useful to people

people. Recombinant DNA technology has resulted in a
boom in biotechnology.

Expression vectors allow a transgene to be expressed in a host cell.

Recombinant DNA techniques have been used to make medically useful proteins.