Tom Strachan • Andrew Read

Human Molecular Genetics

Fourth Edition

Chapter 6
Amplifying DNA:
Cell-based DNA Cloning and PCR
Copyright © Garland Science
1 2011
 Key Points
• In cell-based DNA cloning, target DNA is
fractionated by transfer into bacterial or
yeast cells (transformation).

• Each transformed cell typically takes up

just one target DNA molecule and replicates
it using the host cell’s DNA polymerase.

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 Key Points
• Restriction endonucleases cut DNA molecules at
defined short recognition sequences and are
needed to prepare target and vector DNA for
joining together.

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 Key Points
• Cell-based DNA cloning is slow and laborious.

• However, it can produce very large amounts of

cloned DNA and is the first choice for cloning
large DNA sequences, expressing genes, and
making comprehensive sets of DNA clones
(DNA libraries).

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 Key Points
• DNA cloning can also be performed rapidly in
vitro with the polymerase chain reaction (PCR).

• A purified DNA polymerase is usually used to

replicate a specific DNA sequence selectively
within a complex sample DNA.

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 Key Points
• PCR is also widely used as an assay to quantitate
DNA, and RNA after it has been converted by
reverse transcriptase to complementary DNA
(cDNA).

• Real-time PCR is the most efficient method, and

quantitates PCR products while the PCR reaction
is occurring.
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 DNA cloning
DNA sequences of interest are selectively
replicated
- to produce very large numbers of identical
copies (clones).

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 Molecular hybridization
• The fragment of interest is not amplified or
purified in any way.
• Instead, it is specifically detected within a
complex mixture of many different
sequences.

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 Cell-based DNA cloning
• Developed in the early 1970s
• Uses cells to fractionate complex sample
DNA.
• Idea:
– cut the sample DNA into small pieces
– transport these fragments into suitable bacterial
or yeast cells.

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 HOW TO CUT?

Restriction Endonucleases

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 Restriction Endonucleases
• Cell based DNA cloning is made possible by the
discovery of type II restriction endonucleases.

• Restriction endonucleases serve to protect

bacteria from invading pathogens, notably
viruses.

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 Restriction Endonucleases
• After recognizing specific short sequence
elements in the foreign DNA, the restriction
endonucleases cleave the foreign DNA in the
vicinity of each such element.

• The bacterial cell DNA is left uncut because it

is protected by DNA methylation.

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 Restriction Endonucleases
• Different restriction endonucleases
recognize different specific sequences.

• We can utilize this in order to make cuts in

the DNA regions that we want to target.

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Restriction Endonucleases

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Restriction Endonucleases

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http://rebase.neb.com
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 Transformation
• (definition used in this context)
‘Uptake by a competent bacterial cell of
naked high molecular weight DNA from the
environment.’

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 Transformation

Usually each host cell takes up only one type

of recombinant DNA.

As a result we have recombinant cells.

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 Transformation
• Cells grow and multiply by repeated cell
division.

• Recombinant DNA typically replicates

independently of the host cells
chromosomes.

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• Each recombinant cell may have a different
DNA sequence picked up. How can we
separate these cells from each other?

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• The goal in plating out is to get colonies
arising from single cells.

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 Manageable pieces of target DNA are joined to
vector molecules by using restriction
endonucleases and DNA ligase
• There are different classes of restriction
enzymes/endonucleases
– See Box 6.1

– Type II restriction endonucleases have defined

positions and produce defined restriction
fragments.
– Many type II R.E.s recognize palindromic
regions.
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The final product of the R.E. digestion can be
with 5’ / 3’ overhangs or with blunt ends

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Asymmetric cutting of a
palindromic sequence
generates restriction
fragments with two
overhanging ends that
are identical in sequence
and at the same time
complementary in base
sequence.

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They will have a tendency to
associate with each other (or
with any other similarly
complementary overhang)
by forming base pairs.

Such overhanging ends are

thus often described as sticky
ends.

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Type II restriction endonucleases also allow for artificial
recombination of DNA molecules.

By cutting a vector molecule and the target DNA with restriction

endonuclease(s) producing the same type of sticky end, vector–
target association is promoted by base pairing between their sticky
ends.

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Usually ligation reactions
are designed to promote
the joining of target DNA
to vector DNA and to
minimize vector
cyclization and other
unwanted products such as
linear concatemers
produced by sequential
end-to-end joining.
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Basic DNA cloning in bacterial cells uses vectors based
on naturally occurring extrachromosomal replicons

• Most cell-based DNA cloning uses modified

bacteria as the host cells.

– Short cell cycle times

– Rapid multiplication
– Single circular double stranded chromosome
– Single origin or replication

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Basic DNA cloning in bacterial cells uses vectors based
on naturally occurring extrachromosomal replicons

Usually, the target DNA fragments do not have a

functional origin of replication.

They need to be attached to a DNA replicon.

This is also known as the vector.

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Basic DNA cloning in bacterial cells uses vectors based
on naturally occurring extrachromosomal replicons

• The extrachromosomal replicons that are found

in bacterial cells usually belong to one of two
classes:
– plasmids and
– bacteriophages (phages).

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Plasmids
• Non-essential DNA molecules that replicate
independently of the host cell’s chromosome.

• Vertically distributed to daughter cells after

division of the host cell,

• But can be transferred horizontally to neighboring

cells during bacterial conjugation events.
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Plasmids
• Often have small circular double-stranded DNA
molecules,

• Some plasmids have linear DNA.

• They range in size from 2 kb to more than 200 kb

and individually contain very few genes.

• Copy numbers are also highly variable…

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Plasmids

Different plasmids can coexist in a cell.

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Plasmids

• Natural examples of bacterial plasmids include

plasmids that carry the sex factor (F) and those
that carry drug-resistance genes.

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Plasmids
• Some plasmids sometimes insert their DNA into
the bacterial chromosome (integration).

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Plasmids
• Some plasmids sometimes insert their DNA into
the bacterial chromosome (integration).

• Such plasmids, which can exist in two forms—

extrachromosomal replicons or integrated
plasmids—are known as episomes.

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• If the plasmid can have drug resistance
genes on it.

– Is there a way of utilizing these genes for our

benefit in DNA cloning?

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Phages

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Phages

• A bacteriophage (or phage, for short) is a virus

that infects bacterial cells.
• Unlike plasmids, phages can exist extracellularly
but, like other viruses, they must invade a host cell
to reproduce.
• They can only infect and reproduce within certain
types of host cell.

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Phages

• Phages may have DNA or RNA genomes

• In DNA phages the genome may consist of
double-stranded DNA or single-stranded DNA
may be linear or circular.
• Considerable variation in genome sizes, from a
few kilobases to a few hundred kilobases.

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Phages

• Like episomal plasmids, some bacteriophages,

such as phage λ, can also integrate into the host
chromosome.

• In here, the integrated phage sequence is known

as a prophage

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