Chemical Synthesis,
Sequencing, and
Amplification of DNA

Chemical Synthesis of DNA
1.
2.
The Phosphoramidite method
Uses of synthesized oligonucleotides

Fig 5.1

Fig 5.2

Fig 5.3

Fig 5.4

Fig 5.5

Fig 5.6

Fig 5.7

Fig 5.8

Fig 5.9

Fig 5.10

Fig 5.11

Fig 5.12

Fig 5.13

table 5.1

DNA Sequencing Techniques
2.
3.
1.
4.
Dideoxynucleotide procedure for sequencing DNA
Automated DNA sequencing
Using bacteriophage M13 as a DNA sequencing vector
Primer walking

Dideoxynucleotide

Cycle Sequencing Reaction
* Templates
* Primers
* AmpliTaq DNA Polymerase, FS
* BigDye Terminators
* Others (Buffer, Mg2+ , dNTP…)

Templates
1.
2.
3.
Double Stranded Plasmid DNA
PCR Product
Single Stranded DNA

Templates: Quality Requirement
Plasmid DNA:

RNA, Genomic DNA and Protein free

Recommended Purification Method



QIAGEN DNA Isolation Products
Alkaline lysis with PEG PPT
PCR Product:
 if single band produced: remove excess primers, dNTP if multiple band produced: gel purification followed by extraction protocol is required

Primers
•
•
•
•
•
Length: generally 18-24 bases
Specific Hybridization Site
Avoid Secondary Structure
Avoid Strings of 3 or More of One Base in the Primer Sequence
High Purity

Sequencing Reaction ( standard protocol)
Component Quantity
Plasmid DNA 0.2-0.5 m g
Primer 3.2 pmole
BigDye Terminator Kit 8 m
L
Deionized water q.s.
Total Volume 20 m
L
*Adjust the concentration of template and primer to make the total volume of template and primer < 12 m
L

PCR Product Required for Cycle Sequencing
Reaction
Size(bp) Template quantity(ng)
100-200 1-3
200-500 3-10
500-1000 5-20
1000-2000 10-40
>2000 40-100

Cycle Sequencing Program
 96 ℃ , 2 min
 96 ℃ , 10 sec
 50 ℃ , 5 sec 25 cycle
 60 ℃ , 4 min

Recommended Alcohol
Precipitation Method Protocol
• Add the ethanol/EDTA solution to each well:
5.0 uL of 125mM EDTA
60 uL of 100% ethanol
•Mix by inverting 4 times than leave the tubes at room temperature for
15 minutes to precipitate the extension products
• Spin the tubes for 20 minutes at maximum speed in a microcentrifuge
( Proceed to the next step immediately)
• Carefully aspirate the supernatants completely
• Add 60 uL of 70% ethanol to the tubes and mix briefly
• Spin the tubes for 5 minutes at maximum speed (at 4ºC)
• Aspirate the supernatants completely
• Dry the samples in a vacuum centrifuge for 5-10 minutes

Next-Generation Sequencing

高通量定序 (High -Throughput
Sequencing, HTS)

合成定序法 (Sequencing by Synthesis,
SBS)

大量同步定序法 (Massive Parallel
Sequencing, MPS)

Next Generation Sequencing

Applied Biosystems/Agencourt (SOLiD)