PREFI LEC: DNA SEQUENCING Flashcards
Refers to the order of the nucleotides in the DNA molecule
DNA SEQUENCE
Applications of DNA sequencing in medical laboratory:
1. Detection of mutation 2. Typing microorganisms 3. Identifying human haplotypes 4. Designating polymorphisms 5. Treatment strategies
DNA SEQUENCE
Sequencing Methods:
- Direct sequencing: manual and automated
- Pyrosequencing
- Bisulfite DNA sequencing
- RNA sequencing
- Next-Generation sequencing
Direct determination of the order, or sequence of nucleotides in a DNA polymer
DIRECT SEQUENCING
Most specific & direct method for identifying genetic lesions (mutations)/ polymorphisms
DIRECT SEQUENCING
2 TYPES OF DIRECT SEQUENCING
- Manual sequencing (chemical/ MaxamGilbert & Sanger sequencing)
- Automated fluorescent sequencing (dye primer & dye terminator sequencing)
2 PROCESS IN MANUAL DNA SEQUENCING
- Chemical (Maxam-Gilbert) Sequencing
- Dideoxy Chain Termination (Sanger) Sequencing
Requires a ds/ss version of the DNA region to be sequenced, with 1 end radioactively labeled (32P)
- Chemical (Maxam-Gilbert) Sequencing
Allan M. Maxam & Walter Gilbert
Chemical (Maxam-Gilbert) Sequencing
Sequencing proceeds in 4 separate reactions
Chemical (Maxam-Gilbert) Sequencing
Template used in Chemical (Maxam-Gilbert) Sequencing
labeled fragment
Addition of a ______ = ssDNA would break at specific nucleotides
strong reducing agent (10% piperidine)
After reactions: fragments will be separated by size on a ______
denaturing polyacrylamide gel (6-20%)
Short fragments (up to 50bp) =
1-2 hours
Long fragments (>150 bp) =
7-8 hours
Frederick Sanger
Uses dideoxynucleotides (ddNTPs) to determine the order/sequence of nucleotides in a nucleic acid
Primer complementary to DNA to be sequenced
Dideoxy Chain Termination (Sanger) Sequencing
Product detection of sequencing of Dideoxy Chain Termination (Sanger) Sequencing
- Primer is attached at the 5’ end to a 32P/fluorescent dye-labeled nucleotide
- Incorporate 32P/35S-labeled dNTPs in the nucleotide sequencing reaction mix (internal labeling)
are added, terminating the DNA synthesis (chain termination)
ddNTPs
5’-3’ phosphodiester bond cannot be established to incorporate a subsequent nucleotide
Lack OH
Components: Mixed in 4 reaction tubes
- DNA template (PCR product)
- Radioactively-labeled primer
- Enzyme (DNA polymerase)
- dNTPs (all 4)
- Buffer (20 mM EDTA, formamide, gel tracking/loading dyes)
- Different ddNTPs in each of the 4 tubes
Sequencing reaction of Dideoxy Chain Termination (Sanger) Sequencing
thermal cycler (cycler sequencing)
Automated reading of DNA sequence ladder requires fluorescent dyes (4 distinct colors) to label primers / sequencing events
1. Fluorescein
2. Rhodamine
3. Bodipy (4,4-difluoro-4-bora-3a,4a-diazas-indacene)
AUTOMATED FLUORESCENT SEQUENCING
Fluorescent dyes can be distinguished by
automated sequencers
Approaches (to label fragments according to their terminal ddNTP):
dye primer & dye terminator sequencing
Fragments ending in ddATP, read as A in the sequence =
green dye
Fragments ending in ddCTP, read as C in the sequence =
blue dye
Fragments ending in ddGTP, read as G in the sequence =
black/yellow dye
Fragments ending in ddTTP, read as C in the sequence =
red dye
4 different fluorescent dyes are attached to 4 separate aliquots of the sample
Dye Primer Sequencing
Dye molecules are attached to the 5’ end of the primer = 4 versions of the same primer w/ different dye labels
Dye Primer Sequencing