DNA Sequencing

Principle of Sanger Sequencing: Chain Extension

• Design a WHAT, WHAT to the template strand

• Mix the primer with your WHAT

• Raise the temperature until the DNA WHAT (H bonds between strands broken)

• As temperature is lowered, primers WHAT to the template

• Extend primer using WHAT activity to WHAT DNA

Principle of Sanger Sequencing: Chain Extension

• Design a PRIMER, COMPLEMENTARY to the template strand

• Mix the primer with your DNA

• Raise the temperature until the DNA DENATURES (H bonds between strands broken)

• As temperature is lowered, primers ANNEAL to the template

• Extend primer using POLYMERASE activity to SEQUENCE DNA

Sanger Sequencing AKA Dideocy Sequencing

• ddNTP = WHAT

• dNTP = WHAT

Sanger Sequencing AKA Dideocy Sequencing

• ddNTP = Dideoxyribonucleotide triphosphate

• dNTP = Deoxyribonucleotide triphosphate

Sanger Sequencing: Original Method - All reactions contain

• WHAT

• WHAT

• WHAT

• WHAT

Sanger Sequencing: Original Method - All reactions contain

• DNA Template

• DNA Polymerase

• Primers (lots of copies)

• dNTPS (A, T, C, G)

Sanger Sequencing: Original Method - in the four separate reaction

• WHAT

• WHAT

• WHAT

• WHAT

Sanger Sequencing: Original Method - in the four separate reaction

• ddATP

• ddTTP

• ddCTP

• ddGTP

Sanger Sequencing: Original Method

When dNTP binds open -OH

• Another open WHAT is added at WHAT

• WHATis continuous

Sanger Sequencing: Original Method

When dNTP binds open -OH

• Another open -OH is added at 3’

• CHAIN EXTENSION continuous

Sanger Sequencing: Original Method

When ddTTP binds to open -OH

• WHAT is added at 3’

• DNAP cannot add any more WHAT

• Chain WHAT

Result: Set of nested fragments of every possible length with a WHAT

Sanger Sequencing: Original Method

When ddTTP binds to open -OH

• A-H is added at 3’

• DNAP cannot add any more NTPs

• Chain TERMINATES

Result: Set of nested fragments of every possible length with a 3’ ddTTP

Sanger Sequencing: Original Method

• You then analyze each reaction in a different lane of a WHAT to determine the WHAT

• Special gel that can resolve 1 bp WHAT (WHAT)

Sanger Sequencing: Original Method

• You then analyze each reaction in a different lane of a GEL to determine the SEQUENCE

• Special gel that can resolve 1 bp DIFFERENCES (POLYACRYLAMIDE)

Sanger Sequencing: Modern Method

• In modern Sanger sequencing, the ddNTPs are each WHAT tagged

• The reaction occurs in a WHAT with all the WHAT

• The result is then run on a single lane where nucleotides are identified based on the WHAT tag

• The process is WHAT

Sanger Sequencing: Modern Method

• In modern Sanger sequencing, the ddNTPs are each FLUORECENTLY tagged

• The reaction occurs in a SINGLE TUBE with all the ddNTPs

• The result is then run on a single lane where nucleotides are identified based on the ddNTP tag

• The process is AUTOMATED

Benefits of Sanger sequencing:

• Highest WHAT of sequencing methods

• For many smaller applications, it’s WHAT and practical

Benefits of Sanger sequencing:

• Highest FIDELITY of sequencing methods

• For many smaller applications, it’s COST-EFFECTIVE and practical

Issues with Sanger sequencing:

• Sequencing on a genomic scale is WHAT and WHAT

• Reads max. WHAT bp long

Issues with Sanger sequencing:

• Sequencing on a genomic scale is SLOW and EXPENSIVE

• Reads max. 900 bp long

Next-generation sequencing

Massively parallel sequencing, where the whole genome is broken into fragments and sequenced at once

Benefits of next-gen sequencing:

• High WHAT of reads (millions compared to <100 for Sanger)

• Much WHAT per-bp cost

Benefits of next-gen sequencing:

• High VOLUME of reads (millions compared to <100 for Sanger)

• Much LOWER per-bp cost

Issues with next-gen sequencing:

• Higher WHAT rates (less fidelity) compared to Sanger sequencing

• High DNA WHAT and WHAT needed

• WHAT reads (99 – 1000 bp)

Issues with next-gen sequencing:

• Higher ERROR rates (less fidelity) compared to Sanger sequencing

• High DNA CONCENTRATION and PURITY needed

• SHORT reads (99 – 1000 bp)

Third-generation sequencing

Long-read or whole molecule DNA sequencing

Benefits of third-gen sequencing:

• Generates WHAT reads (10 kb – 4 Mb!)

• Makes genome assembly WHAT

Benefits of third-gen sequencing:

• Generates ULTRA-LONG reads (10 kb – 4 Mb!)

• Makes genome assembly STRAIGHTFORWARD

Issues with third-gen sequencing:

• Higher WHAT (but improving)

• Needs careful DNA WHAT and WHAT prep

• More WHAT per-bp compared to next-gen

Issues with third-gen sequencing:

• Higher ERROR (but improving)

• Needs careful DNA EXTRACTION and LIBRARY prep

• More EXPENSIVE per-bp compared to next-gen