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Introductory
Molecular Biology

Areas covered
• Basics
– What info do we need to get started in our
understanding

• Basic techniques
–
–
–
–

Gene cloning
Restriction digestion
Polymerase chain reaction
DNA sequencing

• More advanced techniques
– Genomics Later lecture
– Proteomics Later lecture

What is the point of Mol Biol
• Study of gene regulation
• Ability to isolate one gene from an
organism for study
...
coli, easiest to use and very common
...
coli cell
– Called transformation

• Plate on media
• Select for clones

Techniques to isolate
gene
• Restriction digestion
– Restriction endonucleases
– Enzymes with recognise
specific DNA sequence
– Bind to it and cut it
Here is what happens

This is a schematic of
how the fragments
look on an
agarose gel

Template
DNA

Cool temp

More common to use
PCR

• Polymerase chain reaction
• Cyclical process of DNA synthesis
targeting a specific region of DNA
– DNA pol are special ones from
thermophilic organisms
...
coli
• Uptake of DNA by E
...
coli containing
the correct plasmid
• Induce the culture (add
chemical)
• Check for protein
production on an SDSLane 1 Size standards
PAGE (later lecture)
Lane 2 Control E
...


– Sanger, chain termination method
• Method based on interrupting DNA synthesis in a
specific manner
...
5’
TTGT -OH
PRIMER

3’

POLYMERASE

GA G
AT CTC

How do we sequence DNA?
•
•
•
•
•

Template
Primer
dNTP’s
Polymerase
Buffer
UNKNOWN SEQUENCE

3’
5’

AACAGCTT CAGT…
...
3’
PRIMER

But this is only copying the template,
not giving information on sequence

Little bit of chemistry
• DNA synthesis
• Events are catalysed
by enzyme
• Nucleophilic attack by
O of 3’ –OH group on
Phosphate
• New bond formed
• PPi released
• Next base added

Stopping synthesis
• Chemically modified
nucleotide building
block
• No 3’ –OH group
• Nucleotide can be
added to growing
strand
• But the next base
can not link
• Chain growth
terminates

Controlled interruption of chain
elongation
• DYE-PRIMER method
• Label the primer
– Usually a fluorescent dye

• Set up 4 synthesis reactions
– One for each base

• Spike the A reaction with a low
concentration of ddATP etc
• Separate each reaction mix on
a polyacrylamide gel
• Scan the 4 lanes with a laser
scanner
• Read the order of the bases
from the location of the bands

• DYE-TERMINATOR method
• Dye is on the ddNTP
• Each ddNTP has a unique dye which fluoresces
at a distinct wavelength
• Reaction can take place in one tube with spiked
with 4 dye-labelled ddNTPs
• Reaction separated by capillary gel
electrophoresis
• Scan the fragments by colour as they emerge
from capillary
• Multi-coloured chromatogram, each colour
represents a base
• Read off sequence from the order of the peaks
5’

3’

Limitation
• Usual output from a single reaction approx
800-1000 bp of information
• What if you are trying to sequence
something bigger?
• Genomics in separate lecture



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