Molecular Approaches to Nutrition

1
Molecular Approaches to Nutrition

• Molecular Biology 2
• Principles and Methods Dr. Janice Drew

2
Principles and Methods

• Purification and handling of DNA/RNA
• Gel Electrophoresis
• Nucleic Acid Hybridisation
• Cutting and rejoining DNA
• Methods of introducing DNA into cells
• PCR
• DNA sequencing
• Sequence interpretation

3
Handling of DNA/RNA

• DNases and RNases
• Glass and plasticware
• Solutions

4
Extraction of DNA/RNA

• DNA extraction
• Alkaline lysis
• Neutralisation
• Precipitation of proteins and cell debris
• Precipitation or elution using spin column

• RNA extraction
• Lysis incorporating instantaneous inactivation of
  RNases
• Separation of contaminating DNA
• Precipitation or elution using spin column

5
Quantitation and analysis of DNA/RNA

• Spectrometric determination at 260nm
• Gel Electrophoresis
• Agilent technology

6
Gel Electrophoresis

• Nucleic acids are negatively charged
• PO4- groups
• Electrophoresis resolves by size
• Agarose is the usual gel matrix
• Ethidium bromide/SYBR green stains DNA RNA
• Fluorescent colour under UV illumination

7
Agarose Gel Preparation
Agarose fine white powder polysaccharide
(galactose polymer) isolated from seaweed. 1
(w/v) dissolves in Tris-acetate buffer at 60 C
and the solution sets at 30 C
8
Agarose Gel Image
9
Agilent Technology
10
Electropherogram showing Agilent analysis of
total RNA
28S
18S
Fluorescence
Times (seconds)
11
Hybridisation - Identification of DNA/RNA

• Agarose gel electrophoresis separates nucleic
  acids on the basis of size - does not identify
  DNA/RNA fragments
• Nucleic acid probes are used to identify specific
  DNA/RNA sequences in a gel
• Probe is a known nucleic acid sequence
• Relies on the principle of base pairing -
  complementary DNA/RNA sequences stick (hybridise)
  together

12
Hybridisation - Identification of DNA/RNA

• Many molecular biology procedures to identify
  specific DNA/RNA sequences use this principle -
• Southern (DNA) or Northern (RNA) blotting
• In situ hybridisation
• Microarrays
• Antisense technologies

13
Probe Production

• Synthesise a known fragment
• OR
• Purify a known fragment of DNA
• Restriction enzyme digestion
• Heat denature to give single strands
• Add primers, DNA polymerase and
  radioactive/colour labelled nucleotides
• Make a radioactive/ colour labelled complementary
  strand
• Denature to give single strands

14

nylon membrane and transferred DNA
TREAT and BLOT GEL Transfer to nylon membrane
HYBRIDISATION OVEN Incubate filter and probe
in hybridisation buffer
Southern/Northern Blotting and Hybridisation

15
Restriction Endonucleases

• Restriction endonucleases cut DNA
• Present in bacteria
• Cut at sequence specific sites
• Usually 4 or 6 base pairs long
• Bacteria use them to destroy foreign DNA
• Bacteria protect their own DNA against
  self-cutting by special methylation of their DNA
• Restriction enzymes can be purified and are used
  in genetic engineering studies

16
Restriction Endonucleases

• Example Restriction enzymes
• EcoR I (E. coli Restriction Endonuclease I)
• Stu I (Streptomyces tubercidicus I)

Stu I
EcoR I
Palindromic Axis of rotational symmetry
AGGCCT TCCGGA
5
3
GAATTC CTTAAG
5
3
5
3
5
3
Sticky Ended Blunt Ended
17
Molecular Scissors and Glue

• There are 100s of restriction enzymes, each one
  with a different recognition site
• These enzymes are molecular scissors and can be
  used to specifically cut long DNA strands into
  smaller pieces
• The T4 virus, which infects E. coli, has an
  enzyme, T4 DNA ligase, which can form a
  phosphodiester bonds between DNA molecules
• Purified T4 DNA ligase can be used as molecular
  glue to join pieces of DNA. This enzyme is
  widely used for DNA cloning

18
Ligation of DNA
T4 DNA Ligase
Stu I
EcoR I
OH 3 5 PO4
PO4 5 3OH
T4 DNA Ligase
T4 DNA ligase catalyses the formation of
phosphodiester bonds
19
Methods of introducing DNA into cells

• Plasmids
• Viruses
• DNA and RNA viruses
• Phage vectors

20
Cloning DNA into Plasmids

• Bacteria have a circular DNA genome
• 5 to 10 million base pairs (bp) in size
• Many bacteria also contain plasmids
• Small circular DNA molecules, 3,000 to 50,000 bp
• Note The bacterial genome is not a plasmid
• Plasmids contain extra genes which are often
  vital for the survival of the bacterium
• Nutrient metabolism, antibiotic resistance
• Plasmids can be used as vectors in which foreign
  DNA can be ligated (cloned)

21
A General Laboratory Plasmid
Multiple Cloning Site
A foreign gene can be ligated into a plasmid, and
the genetically engineered plasmid introduced
into E. coli.
22
Cloning DNA into a Plasmid
Both plasmid and foreign DNA have sticky EcoR I
ends
Agar plates contain antibiotic. Grow at 37 C
Insertion into E. coli (transformation)
Place 1 colony in liquid media
antibiotic. Grow at 37 C
Purify Plasmid DNA (Billions of copies)
23
DNA and Retroviruses can serve as vehicles for
the introduction of new DNA into a cell
24
DNA / RNA viruses as vehicles
gene x
Viral DNA
Chromosomal DNA
Integration into genome
Gene Therapy and Transgenics
25
Polymerase Chain Reaction (PCR)

• PCR generates multiple copies of DNA
• Heat resistant DNA polymerase used to copy a
  section of DNA e.g Taq
• Very efficient copying
• Billions of copies from a single template DNA
• Small volume / quick analysis

26
Polymerase Chain Reaction (PCR)

• Entire reaction performed in single tube
• 10 to 50 µl volume
• Reaction contains
• Template DNA, heat resistant DNA polymerase, a
  pair of specific DNA primers (in excess over the
  template), nucleotide bases, appropriate reaction
  buffer
• Reaction is repeatedly cycled through 3
  temperatures (x30)
• 95 C (makes DNA single stranded)
• 55 - 60 C (primers anneal to template DNA)
• 72 C DNA polymerase copies DNA, starting from
  the primers

27
A Thermocycler
This thermocycler can accept 1500 reactions at a
time, and complete them in 2 to 4 hours.

28
Principal of PCR
DNA (Double Stranded)
5

3

A
G
C
T
A
G
C
A
T
G
T
T
G
C
G
C
G
T
A
T
C
A
T
G
T
A
C
A
G
T
G
C
A
T
A
C
G
T
C
C
C
C
T
T
A
G
C
T
































































































T
C
G
A
T
C
G
T
A
C
A
A
C
G
C
G
C
A
T
A
G
T
A
C
A
T
G
T
C
A
C
G
T
A
T
G
C
A
G
G
G
G
A
A
T
C
G
A
3

5

Heat Denature (Becomes Single Stranded)
5

3

A
G
C
T
A
G
C
A
T
G
T
T
G
C
G
C
G
T
A
T
C
A
T
G
T
A
C
A
G
T
G
C
A
T
A
C
G
T
C
C
C
C
T
T
A
G
C
T
































































































T
Heat to 72 C
Cool. This allows specific primers to anneal as
shown
o
o
l

t
o

5
5

C
Heat to 72 C. Heat resistant DNA polymerase
extends new DNA from the primers
29
DNA Sequencing

• A specific primer binds to denatured DNA
• Heat resistant DNA polymerase extends a new
  strand from this primer
• Complementary nucleotides are added as
  appropriate
• In the reaction are small quantities of coloured
  dideoxynucleotides
• Colours ddTTP ddGTP ddATP ddCTP
• These prevent further additions (terminators)

30
Dideoxynucleotides
ddNTPs have no 3 OH, so when added they cannot
form the phosphodiester bond required to add the
next nucleotide
31
DNA Sequencing Reaction

• The reaction is boiled to make all the DNA
  single stranded and then the reaction is resolved
  on a long polyacrylamide or capillary gel in a
  DNA sequencer

32
Electropherogram of sequencing gel
33
Decoding DNA sequence data
34
(No Transcript)