Clase Secuenciación clásica

1. DNA Sequencing

2. DNA sequencing
 Determination of nucleotide sequence
 the determination of the precise linear order
(Physical mapping) of nucleotides in a sample of
DNA
 Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
 They depend on the production of a mixture of
oligonucleotides labeled either radioactively or fluorescein,
with one common end and differing in length by a single
nucleotide at the other end
 This mixture of oligonucleotides is separated by high
resolution electrophoresis on polyacrilamide gels and the
position of the bands determined

3. Maxam-Gilbert
• Walter Gilbert
– Harvard physicist
– Knew James
Watson
– Became intrigued
with the biological
side
– Became a
biophysicist
• Allan Maxam

4. The Maxam-Gilbert
Technique
• Principle - Chemical
Degradation of Purines
– Purines (A, G) damaged by
dimethylsulfate
– Methylation of base
– Heat releases base
– Alkali cleaves G
– Dilute acid cleave A>G

5. Maxam-Gilbert
Technique
• Principle
Chemical Degradation
of Pyrimidines
– Pyrimidines (C, T) are
damaged by hydrazine
– Piperidine cleaves the
backbone
– 2 M NaCl inhibits the
reaction with T

6. Maxam and Gilbert Method
Chemical degradation of purified fragments (chemical degradation)
The single stranded DNA fragment to be sequenced is end-labeled by
treatment with alkaline phosphatase to remove the 5’phosphate
It is then followed by reaction with P-labeled ATP in the presence of
polynucleotide kinase, which attaches P labeled to the 5’terminal
The labeled DNA fragment is then divided into four aliquots, each of which is
treated with a reagent which modifies a specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue
2. Aliquot B + formic acid, which modifies adenine and guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific
for cytosine
The four are incubated with piperidine which cleaves the sugar phosphate
backbone of DNA next to the residue that has been modified

7. Maxam-Gilbert
sequencing - modifications

8. Maxam-Gilbert sequencing - summary

9. Advantages/disadvantages
Maxam-Gilbert sequencing
 Requires lots of purified DNA, and many intermediate
purification steps
 Relatively short readings
 Automation not available (sequencers)
 Remaining use for ‘footprinting’ (partial protection against
DNA modification when proteins bind to specific regions, and
that produce ‘holes’ in the sequence ladder)
In contrast, the Sanger sequencing methodology
requires little if any DNA purification, no restriction
digests, and no labeling of the DNA sequencing
template

10. Sanger Method
 Fred Sanger, 1958
– Was originally a
protein chemist
– Made his first mark
in sequencing
proteins
– Made his second
mark in sequencing
RNA
 1980 dideoxy
sequencing

11. Original Sanger Method
 Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNA
 Requires DNA polymerase I
 Requires a cloning vector with initial primer
(M13, high yield bacteriophage, modified by
adding: beta-galactosidase screening,
polylinker)
 Uses 32P-deoxynucleoside triphosphates

12. Sanger Method
 in-vitro DNA synthesis using ‘terminators’, use of dideoxi-
nucleotides that do not permit chain elongation after their
integration
 DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
 Requires a primer, DNA polymerase, a template, a mixture
of nucleotides, and detection system
 Incorporation of di-deoxynucleotides into growing strand
terminates synthesis
 Synthesized strand sizes are determined for each di-
deoxynucleotide by using gel or capillary electrophoresis
 Enzymatic methods

13. Dideoxynucleotide
no hydroxyl group at 3’ end
prevents strand extension
CH2
O
O
PPP
5’
3’
BASE

14. The principles
• Partial copies of DNA fragments made
with DNA polymerase
• Collection of DNA fragments that
terminate with A,C,G or T using ddNTP
• Separate by gel electrophoresis
• Read DNA sequence

15. CCGTAC
3’ 5’
5’ 3’
primer
dNTP
ddATP
GGCA
ddTTP
GGCAT
ddCTP
GGC G
ddGTP
GG
GGCATG
A T C G

16. Dideoxy Chain Terminator
Template
Primer
Extension Chemistry
– polymerase
– termination
– labeling
Separation
Detection

17. Chain Terminator Basics
Target
Template-Primer
Extend
ddA
ddG
ddC
ddT
Labeled Terminators
ddA
AddC
AC ddG
ACG ddT
TGCA
dN : ddN
100 : 1

19. Electrophoresis

20. Sanger Method Sequencing Gel

21. Template
ssDNA vectors
– M13
– pUC
PCR
dsDNA (+/- PCR)

22. Primers
Universal primers
– cheap, reliable, easy, fast, parallel
– BULK sequencing
Custom primers
– expensive, slow, one-at-a-time
– ADAPTABLE

23. Extension
Polymerase
– Sequenase
– Thermostable (Cycle Sequencing)
Terminators
– Dye labels (“Big Dye”)
• spectrally different, high fluorescence
– ddA,C,G,T with primer labels

24. Separation
Gel Electrophoresis
Capillary
Electrophoresis
– suited to automation
• rapid (2 hrs vs 12 hrs)
• re-usable
• simple temperature
control
• 96 well format

25. Sample Output
1 lane

27. Sequencing of DNA by the Sanger method

28. Comparison
• Sanger Method
– Enzymatic
– Requires DNA
synthesis
– Termination of chain
elongation
• Maxam Gilbert Method
– Chemical
– Requires DNA
– Requires long stretches
of DNA
– Breaks DNA at different
nucleotides