Lecture 1

MOLECULAR BIOLOGY

Introduction to Molecular
Biology Techniques
Dr. Jane Sherwood H1.18
Email jms@dmu.ac.uk
I work Mondays Tuesdays and
Thursdays

Introduction to
Molecular Biology
Techniques

What is molecular biology?

What are its uses?
Important background revision
DNA/RNA/protein structure

Textbooks
Essential Cell Biology (ECB)
Chapter 10
Alberts et al.
Published by Garland

Textbooks

Any other up-to-date (since 2009)

molecular biology text book

Areas to be covered during

the next 4 weeks (plus 4
weeks in 2016)
Purification of nucleic acids
Manipulating DNA
PCR including qPCR
Sequencing of DNA Sanger method
and new generation sequencing
Southern (Dot blots) and Northern
blotting
Western blotting
FISH and chromosome painting

Learning objectives
To review the structure of DNA
and RNA
To be able to draw flow diagrams
to illustrate the processes of DNA
and RNA purification
To know how to quantify how
much DNA or RNA there is in your
sample

Central Dogma of Molecular

Biology
From Essential
Cell Biology
Alberts et al.

From Essential
Cell Biology
Alberts et al.

From
Essential
Cell Biology
Alberts et al.

Purification of DNA
Many different commercial
kits available
DNA free from RNA and
_________ contamination

Purification/
isolation of
DNA
Pre-kit method

From Principles and techniques of

biochemistry and molecular biology by
Wilson and Walker 7th Ed CUP

Usually use commercially available

kits but...

you need to know the function of each reagent

for troubleshooting when things go wrong!

Wizard genomic DNA purification kit

from whole blood
pure ds DNA
PDF file for instructions

Blood storage prior to

purification of DNA
Can be sent by first class post
Needs to be collected in anti-coagulant
(stops blood clotting), ie. ________,
citrate, heparin
2-8 C for up to 2 months (?yields)

Obtaining the DNA from

the white blood cells
Cell lysis solution

NaOH and SDS (sodium

dodecylsulphate)
Lysis of red cells leaving white cells
intact
Gentle inversion of tube
Centrifuge to remove ______ cell
components - pellet = white cells

Nuclei lysis solution

White cells and their nuclei are lysed
and solubilized
Optional RNase treatment at this stage
(Southern blots)
Debris in ________ - DNA in
supernatant

Purifying the DNA

Protein precipitation

Salt precipitates cellular proteins

leaving high molecular weight genomic
DNA in solution

Isopropanol/ethanol
precipitation

Genomic DNA is concentrated and

desalted

Store DNA at 2-8 C

_________ 5-15g from 300l
whole blood

Add RNase here

From :Wizard Genomic

DNA Purification Kit

Checking the integrity and

quantity of DNA
Gel electrophoresis

Check integrity
Estimate yield (quantity)

Run DNA samples of known concentration and

compared intensity of bands to unknown - rough
estimate only!

DNA determination by
spectrophotometry

Amount of UV absorbed by sample is directly

proportional to amount of DNA
Absorbance260nm of 1.0 = 50 g of ds DNA/mL

RNA

From Essential
Cell Biology
Alberts et al.

Isolation of RNA
What type of RNA?
Total RNA
mRNA

Isolation of RNA
Many different methods
Problem RNases

Present all around particularly on our skin

Take care not to degrade the RNA you have
isolated
All glassware, equipment and water must be
treated with DEPC (diethylpyrocarbonate)
Inhibits RNase __________

Many commercial kits are available to

isolate RNA

 Isolation of
RNA - general
steps involved

From Principles and techniques of

biochemistry and molecular biology by
Wilson and Walker 7th Ed CUP

Total RNA
isolation
The PAXgene Blood RNA System from
PreAnalytiX

From Patient to purified total RNA

consistent results

Isolation of mRNA
using magnetic
particles

 All mRNA molecules have a poly A tail

(polyadenylation)
Poly A tail will hybridize to poly T
oligonucleotide
Attach poly T to biotin (= biotinylated
oligonucleotide)
Biotin will tightly bind to streptavidin which
is used to coat magnetic beads
(Dynabeads)

TTTTTTTTTTT
Poly T tail

TTTTTTTTTTT

3 AAAAAAAAAAAAA

mRNA

Mix with sample (cells/ nuclei broken open)

polyT binds to poly A tail of mRNA
Mix with streptavidin paramagnetic particles
(SA-PMPs)
mRNA now bound to magnetic particles
Use magnet to form pellet
Remove s/n (supernatant)
Wash beads/mRNA with buffer
This washes away impurities leaving _______
mRNA bound to beads - release mRNA from
poly T tail by resuspending pellet in water

Isolation of mRNA
using magnetic
particles

 Advantages:

Quick, pure (no contaminating DNA or

proteins)
Safer than chemically for traditional
methods

Disadvantages:

Expensive, only for mRNA (need poly A

tail)

Kits are also available for total RNA

preparation from various sources

Quantification and
integrity
of
RNARNA
determination by spectrophotometry
Amount of UV absorbed by sample is directly
proportional to amount of RNA
Absorbance260nm of 1.0 = 40 g of ds RNA/mL

RNA integrity

Most common rRNA molecules are 18S and

28S in eukaryotes on electrophoresis these
should appear as discrete bands

Quick quantification of
DNA/RNA/protein
Video

Learning
outcomes
A clear understanding of the
molecular structure of DNA and RNA
What does 5 and 3 end mean and why is
important to carefully label each end?

Be able to discuss the different DNA

and RNA purification methods and the
problems associated with both
Be able to draw flow diagrams

Be able to describe how DNA and

RNA are quantified once they are
purified

DNA structure
key points

RNA structure
key points

DNA/RNA quantification
key points

DNA purification
Flow diagram
Function of all reagents

RNA purification
Flow diagram