part 1: sequencing

DNA sequencing

direct determination of the order of nucleotides in a DNA polymer

most specific and direct method for identifying genetic lesions

two types of sequencing methods develop in the 1970s

Maxam-Gilbert sequencing

Sanger sequencing

Maxam-Gilbert (chemical) sequencing

required ds or ss version of DNA region to be sequenced w/ one end radioactively labeled

this labeled template aliquoted into four tubes

• each tube treated w/ different chemical w/ or w/o high salt

• addition of strong reducing agent, 10% piperidine, the ssDNA would break at specific nucleotides

reading Maxam-Gilbert sequence

lane in which that band appeared identified the nucleotide, reading from bottom of gel to top

Maxam-Gilbert sequencing: chain breaks at G

base modifier: dimethylsulphate (DMS)

methylates G

Maxam-Gilbert sequencing: chain breaks at G + A

base modifier: formic acid (FA)

protenates purines

Maxam-Gilbert sequencing: chain breaks at T + C

base modifier: hydrazine (H)

splits pyrimidine rings

Maxam-Gilbert sequencing: chain breaks at C

base modifier: hydrazine + salt (H + S)

(salt changes specificity)

splits only C rings

dideoxy chain termination (Sanger) sequencing

modification of DNA replication process

short, synthetic, ssDNA primer complementary to sequences just 5’ to region of DNA to be sequenced is used

basis of Sanger sequencing

primer covalently attached to a ³²P-labeled nucleotide or fluorescent dye-labeled nucleotide (visualization)

in vitro DNA replication occurs to make full length copies of DNA template

for sequencing, modified dideoxynucleotide (ddNTP) derivatives are added to the rxn

dideoxynucleotide (ddNTP) derivatives

lack the -OH group on the 3’ ribose, stopping DNA elongation when ddNTP is added

Sanger sequencing procedure

1:1 mix of templates and labeled primer placed into 4 rxn tubes

• each tube has same ingredients for PCR rxn

• mixtures of all four dNTPs, and one of the four ddNTPS (A, T, C, or G) are then added to each tube

• add DNA pol, after 20 min terminate the rxn by adding stop buffer

sets of synthesized fragments are resolved in a gel - sequencing ladder

Sanger sequencing tube components

ddATP + four dNTPs

ddCTP + four dNTPs

etc

what does dideoxynucleotide do?

terminates chain

Sanger sequencing - sequencing ladder

ladder read from bottom to top

smallest fragment represents first nucleotide attached to primer

band formed each interval when elongation stopped, at corresponding ddNPT in tube

automated Sanger sequencing

use fluorescent dyes instead of radioactive label

two approaches: dye primer and dye terminator

label the fragments synthesized during sequencing according to their terminal ddNTP

fragments resolved by capillary electrophoresis

detector will read the fluorescence corresponding to each ddNTP as these pass through capillary

terminal ddATP color

green

terminal ddCTP color

blue

terminal ddGTP

yellow or black

terminal ddTTP color

red

for automated Sanger sequencing, the color of the dye corresponds to what?

the ddNTP that terminated the strand

resolving fragments by capillary electrophoresis during Sanger sequencing

shorter fragments (closer to the primer) will migrate faster than longer ones

detection in automated Sanger sequencing

as fragments pass through capillary, laser excites terminator dye and signal is detected

• software reads or “calls” these bases from smallest fragments to largest

• compiles these signals creating an electropherogram

electropherogram

series of peaks, one for each of the 4 fluorescent dyes

• a chart of light emission over time

how many bp are used to sequence on an electropherogram?

400 - 800 bp

X and Y-axis on electropherogram

Y-axis: intensity

X-axis: scan numbers

electropherogram - interpretation using base calling

process of identification of bases in a sequence by sequencing software

when base call is not clear, letter N will replace the nucleotide

beginning of chart: messy peaks, sequences before 30 bp are not reliable

what do the grey bars on an electropherogram indicate?

Q-value, which indicates the probability of correct call; anything >20 is acceptable

electropherogram - end interpretation at end of run

can get up to 1,000 reliable bases

will see peak quality begin to degrade, Q-value drops below acceptability

electropherogram - DNA contamination

presence of dye blobs - bright flashes of fluorescence that shows as large broad peaks

combination of short and large peaks indicates low DNA concentration and contamination

electropherogram - no DNA at all

no template shows as random noise

peaks are very short (~50 intensity units) indicating that there is no DNA

electropherogram - too much DNA

represented by very high intensity peaks at beginning of run. then, quickly drop

electropherogram - heterozygous mutations

software can compare two sequences to identify mutations

heterozygous mutations are observed as double peaks in affected bp

must be confirmed by running the reverse sequence

electropherogram - heterozygous deletions or insertions

InDels (insertions or deletions) affect all positions of sequence downstream of mutation

overlaid sequences will show minus bases if a deletion

application of automated Sanger sequencing

microbial, plasmid, targeted DNA, mitochondrial, HLA sequencing

validation of sequencing

gene expression analysis