Restriction Digests

(Restriction Fragment Length Polymorphism)

A. Wild type sequence B. Mutant sequence – creation of new restriction

site
RE site

PCR fragment

Digested fragments

C. Mutant sequence – abolition of restriction site

Ladder A B C
Agarose gel Migration of DNA

Step 1. PCR
Step 2. Digest
Step 3. Agarose Gel
 Detecting the Sickle Cell DNA Sequence in the PCR
Product

DdeI Cuts DNA specifically at CTGAG

DdeI Cuts
Normal
YES

Sickle Cell NO
1 2 3 4 5 6 7

308
Dde I Cuts the PCR product 201
S
Normal PCR 107
N
product 88/89
N 49
37
Sickle PCR
product 1. Marker 5. Mother
2. Normal (NN) 6. Father
3. NS control 7. Foetus
4. Sickle (SS) Control
 Amplification Refractory Mutation System
(ARMS)
Forward
primer
(wild type
Wild type sequence
specific) Reverse primer

Forward
Mutant sequence primer X
(mutant X
specific)

Ladder WT MUT

Result = heterozygous

Step 1. PCR
Step 2. Agarose Gel
 Taqman
Step 1 – denature DNA and allow allele-specific probes to anneal.

Mutant probe

Wild type probe

In this case only the mutant probe can bind (DNA only contains the mutant probe sequence and not
the wild type probe sequence).

Key: = fluorophore (e.g. red, green, yellow reporter)

= quencher (if fluorophore is within 10nm it quenches the fluorescence and

so none is released).
 Step 2 – Primers anneal and allow Taq amplification (just like a regular PCR).

Taq

Forward primer

Reverse primer
Step 3 – Taq cleaves fluorophore from probe which is now able to fluoresce.
 Repeat Sizing
Fluorescent tag on primer

Wild type TATATA

Mutant TATATATATATATA

Ladder WT MUT
Step 1. PCR
Step 2. Agarose Gel Well this is the theory! Only problem with
l using agarose gels is the poor resolution.
Resolution between 2 bp differences in PCR
fragment sizes is much better using the
automated DNA sequencer which detects
the fluorescent tag on one of the primers
e.g. for Gilbert’s.
 Differential PCR

1200 bp (wild type

sequence)

600 bp (mutant
sequence)
Ladder WT Het Homo

Step 1. PCR
= deleted in mutant Step 2. Agarose Gel
sequence