Template Preparation for Successful Automated DNA Sequencing
Pure DNA should give an OD260/280 of between 1.7-1.9 (1.5-1.7 is usually OK) and an OD200/260 of about 1.1. Low 260/280 indicates protein contamination, high OD260/280 indicates possible RNA or residual organics contamination. High OD200/260 indicates contamination by organics and/or salts.
Note: Template preparation or purification procedures which involve the use of phenol or chloroform should be avoided if possible. If use of phenol or chloroform can not be avoided an additional ethanol precipitation is recommended.
Commercial Methods for Template Preparation
In addition to the Applied Biosystems Sequencing Chemistry Guide, also see Qiagen’s web page for DNA purification products. Qiagen has an excellent DNA Template Preparation Guide and Troubleshooting publication. Your final purification step should be a column spin using water. Substitute water for “elution” buffer to obtain reproducible results. Elution buffer components (e.g. tris, NaCl, EDTA) cause run failures unless diluted out. Also see "ExoSAP-IT" from USB Corporation for PCR product cleanup.
Ensuring Template Quality
The quality of DNA in a reaction affects the performance of the DNA Analyzer. When preparing DNA templates it is critical to avoid the following:
- Residual salts
- Proteins
- Residual detergents
- Residual RNA
The presence of residual salts, proteins, RNA, and detergents can interfere with capillary electrophoresis and electrokinetic injection. Your current template purification methods may have to be modified to remove residual salts, proteins, and detergents.
Effect of Residual Salts
The 3730 DNA Analyzer is especially susceptible to salt in samples from template preparation. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. If salts and unincorporated dyes are not removed from the
sequencing reaction, they will compete with extension fragments during electrokinetic injection and result in weak signals.
Effect of Proteins
Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array adversely affecting data resolution.
Effect of Residual Detergents
Some methods of template preparation, such as the Thermomax method for M13 preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.
Effect of Residual RNA
Residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.
Template Quantity
Effect of Too Little Template
Too little template or primer reduces the signal strength and peak height. In the worst case, the signal-to-noise level decreases so that bases cannot be called.
Effect of Excess Template
Excess template can affect data quality when present in sample loading onto the DNA Analyzer. Excess template inhibits the injection of extension fragments thus affecting signals generated from the instrument. Excess template can behave similarly to proteins and accumulate in the capillary array, which affects data resolution. (Applied Biosystems 3730/3720xl DNA Analyzers Sequencing Chemistry Guide, p.18-22).
Host Bacterial Strains
The host strain used for a specific template preparation can impact template quality.
Applied Biosystems reports the following on host strain variability vs. sequencing results
- DH5 alpha host strains consistently produce good results.
- HB101, MV1190, JM109 and XL1 Blue host strains show some variability in result quality. XL1 Blue grows slower than most strains and can lead to decreased DNA yields and it does not respond to TB as do other strains (only showing a 2-3 fold increase in cell number per ml).
- JM101 (JM 100 series) is not recommended.
The following is anecdotal:
Avoid Terrific broth and other rich media. Avoid host strains TG1 and TG2 which contain high carbohydrate levels
Some Primer Sequences Used in Fluorescent Sequencing (5’-3’)
Bac Forward...............................................TTTTACTGTTTTCGTAACAGTTTT
Bac Reverse ..............................................CGGATTTCCTTGAAGAGAGTA
Bac(+15)....................................................Reverse ACTTCAAGGAGAATTTCC
Bac1 .........................................................ACCATCTCGCAAATAAATAAG
Bac2..........................................................ACAACGCACAGAATCTAGCG
BacMam 3'..................................................TCCCATATGTCCTTCCGAGTGA
BacMam 5'..................................................ACGTGCTGGTTGTTGTGCTGTCT
BGH Reverse..............................................TAGAAGGCACAGTCGAGG
BK Reverse................................................ACAGGAAACAGCTATGACCTTG
Blue2S.......................................................ACCGCTGCTGCTAAATTCGAA
Bluescript KS..............................................TCGAGGTCGACGGTATC
Bluescript SK..............................................CGCTCTAGAACTAGTGGATC
CBDcenA....................................................TCAACGGCACCACCTGCA
CBDcexLEAD...............................................TAGGTGCAACTGTTGTTCTG
CBDclos.....................................................CAACACCAGTTGTAAATCCA
EBV Reverse..............................................GTGGTTTGTCCAAACTCATC
M13 Forward (-20)......................................GTAAAACGACGGCCAGTG
M13 Forward (-41)......................................GGTTTTCCCAGTCACGAC
M13 Reverse (-27)......................................GGAAACAGCTATGACCATG
M13 Reverse (-48)......................................AGCGGATAACAATTTCACAC
pCDM8 Reverse..........................................TAAGGTTCCTTCACAAAG
pCEP Forward.............................................AGAGCTCGTTTAGTGAACCG
PCMV Forward............................................CGCAAATGGGCGGTAGGCGTG
pTRE 3'......................................................CCACACCTCCCCCTGAAC
pTRE 5'......................................................CGCCTGGAGACGCCATCC
pYESTrp Forward........................................GATGTTAACGATACCAGCC
pYESTrp Reverse........................................GCGTGAATGTAAGCGTGAC
SP6...........................................................TACGATTTAGGTGACACTATAG
T3.............................................................CAATTAACCCTCACTAAAGG
T3 Bluescript..............................................AATTAACCCTCACTAAAGGG
T7.............................................................GTAATACGACTCACTATAGGG
T7 Promoter pET.........................................AAATTAATACGACTCACTATAGGG
T7 Terminator.............................................GCTAGTTATTGCTCAGCGG
T7 Terminator pET.......................................GCTAGTTATTGCTCAGCGG
Vectors Incompatible with the Above Primers
Vector |
Primer(won't work) |
Alternate Primer (will work) |
|---|---|---|
| pT7T3 | T3 |
M13F |
| pT7T3 | T7 |
T7 promoter |
| pSP72 | T7 |
T7 promoter |
| pcDNA | T7 |
T7 promoter |
