1. Molecular Biology lecture -Putnoky
DNS-szekvencia meghatározás
Gilbert
Sanger
(1958)
1980
Molecular Biology lecture -Putnoky

2. Molecular Biology lecture -Putnoky
DNS-polymerases
5'-3' polymerase activity
5'-3' exonuclease activity
3'-5' exonuclease activity
For the synthesis of the new DNA strand are required:
a template DNA strand
a primer (with free 3’ –OH)
deoxynucleoside-triphosphate precursors
dNTP (dATP, dCTP, dGTP, dTTP)
primer
new strand 3’ 5’ 3’ 3’ 5’
template
Molecular Biology lecture -Putnoky

3. Molecular Biology lecture -Putnoky
Sanger sequencing, dideoxy chain termination method
dideoxy ribose
+ labelling α32P-dATP
+ jelölés: pl. α32PdATP
polyacrylamide-urea denaturing gel electrophoresis
+ labelling: α32P-dATP
Synthesis of truncated new stands is accomplished by use of 2,3-dideoxyribonucleoside triphosphates (ddNTPs). These molecules can be incorporated into a growing DNA chain but they cannot form a phosphodiester bond with the next incoming nucleotide triphosphate. Thus the DNA synthesis is terminated resulting in a truncated daughter strand.
This reaction results in many new strands that are labeled and differ in length by one nucleotide. Separation of the truncated strands by gel electrophoresis can then establish the nucleotide sequence of the original DNA fragment.
Molecular Biology lecture -Putnoky

4. Molecular Biology lecture -Putnoky
Polyacrylamide gel electrophoresis
bisAA AA
Polyacrylamide gel electrophoresis (PAGE) is used to separate biological macromolecules, proteins, nucleic acids or polysaccharides according to their electrophoretic mobility.
The gels consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer. Ammonium persulfate and TEMED are added to initiate polymerization in that bisacrylamide molecules form cross-links between two acrylamide molecules.
To separate single stranded DNA or RNA molecules polyacrylamide-urea denaturing gel electrophoresis is applied. To separate protein samples the SDS-PAGE is a very useful method.
Molecular Biology lecture -Putnoky

5. Molecular Biology lecture -Putnoky
Autoradiogam of a sequencing gel
Molecular Biology lecture -Putnoky

6. biológia BSc - Molekuláris biológia előadások - Putnoky
A C G T a a a c c t t c a t a
3-6
biológia BSc - Molekuláris biológia előadások - Putnoky

7. Molecular Biology lecture -Putnoky
Non radioactive labelling with fluorescent dyes
fluoreszcein12- dUTP
Sanger sequencing is the method which prevailed from the 1980s until the mid-2000s. Over that period, great advances were made in the technique, such as fluorescent labelling, capillary electrophoresis, and general automation. These developments allowed much more efficient sequencing, leading to lower costs. The Sanger method, in mass production form, is the technology which produced the first genome sequences.
A novel fluorescent labeling technique made it possible to identify all four dideoxynucleotides in a single lane. Each dNTP is labelled with a different fluorescent dye. In an automated DNA sequencer, the fluorescent dye of each band is activated by a scanning laser at the bottom of the electrophoretic gel. The color of each band is read by a fluorometer, and a computer assembles these as a gel image, which can be read from bottom to top, like a conventional radioactively-labelled sequencing ladder.
Molecular Biology lecture -Putnoky

8. Molecular Biology lecture -Putnoky
Molecular Biology lecture -Putnoky

9. Molecular Biology lecture -Putnoky
Sequence of a DNA fragment or a whole genome can be assembled from several subsequences which originated from separated sequencing reactions (reads). One read contains 500 to 900 nucleotides because of the limits of the sequencing reaction and the separation as well. Overlapping reads can be assembled into one contig by different computer programs.
To estblish the sequence of a DNA fragment at least two reads are necessary. The sequence of both strands should be determined in independent reactions (both strands should be read). In these reaction the „forward” read is the control of the „reverse” read. When the two sequences are identical (reverse complementer sequence is converted by the computer program) the resulted sequence can be accepted as the final sequence.
PRIMER
one read is bp
insert, unknown sequence
Molecular Biology lecture -Putnoky

10. Molecular Biology lecture -Putnoky
The result of a single reaction is a sequencing chromatogram. The rough sequence may need corrections. It is necessary to remove the vector sequences and delete the uncertain reads at the beginning and at the end of the chromatogram.
Molecular Biology lecture -Putnoky

11. Molecular Biology lecture -Putnoky
Molecular Biology lecture -Putnoky

12. Molecular Biology lecture -Putnoky
shotgun sequencing of a genome
Molecular Biology lecture -Putnoky