L6: DNA Sequencing And Synthesis

Question 1

Dideoxy Sequencing of DNA

Answer 1

Procedure devised by Fred Sanger

Involves copying DNA strand to be sequenced using DNA polymerase

Question 2

Properties of DNA polymerase I

Answer 2

Ability to synthesise complementary strand from a ss template

Absolute requirement for a primer with a 3OH group

Ability to incorporate a dideoxynucleotide instead of a deoxynucleotide. Incorporation of dideoxynucleotide -> chain termination (no OH -> cant add nucleotide)

Question 3

Analysis of Reaction Products

Answer 3

Separate fragments through electrophoresis. Separated by mass from ‘sieving’ action of gel

Heat denature to separate DNA strands -> apply to sequencing gel -> run 3-5hr -> autoradiography (expose xray film)

Question 4

Cycle sequencing

Answer 4

Linear amplification sequencing

Denature -> anneal -> primer primes one strand -> taq DNA polymerase -> extend/terminate

Question 5

Automated DNA sequencers - Dideoxy sequencing

Answer 5

Uses cycle sequencing

Use fluorescently-labelled ddNTPs as chain terminators

Sample run on acrylamide/urea gel (or capillary electrophoresis) -> emitted light

Question 6

Helicos Single Molecule Sequencing

Answer 6

Example of next generation sequencing. Involve parallel sequencing of many DNA fragments simultaneously. DNA fragments immobilised on beads or chips

Billions of short sequences read simultaneously

Question 7

Method of Helicos Single Molecule Sequencing

Answer 7

1. Cut genomic DNA into millions of fragments
2. Add oligo dA tail
3. Anneal to chip containing millions of immobilised oligo dT molecules -> holds DNA onto surface of chip
4. Use fluorescent tag and digital imaging system to determine precise location of each molecule

Question 8

Synthesis of Long Oligonucleotides

Answer 8

Length determined by coupling efficiency

To synthesise longer oligonucleotides (and make ds), synthesise 60b oligonucleotides with a 20b overlap

5’ end oligos in excess -> 1 cycle PCR (gaps filled) -> annealing of 5’ end oligos + PCR 25 cycles -> long ds product

Question 9

Uses of Synthetic Oligonucleotides

Answer 9

Synthesis of genes (from joining of oligonucleotides)

PCR primers

Linkers (used to add restriction site to ends of piece of DNA)

Hybridisation probes (e.g in screening DNA libraries or Hybridisation)

Mutagenesis (from imperfect match between oligonucleotide and target gene when gene is copied)

Antisense oligonucleotides used to target mRNA block translation -> gene expression

Affinity chromatography- attached to matrix in column oligonucleotides-> purify specific DNA binding proteins

Preparing DNA microarrays- gene specific sequences arranged on grid, microscope slide or chip. Can be used to analyse gene expression

Question 10

Incorporation of dideoxynucleotide

Answer 10

-> chain termination

no 3’OH -> cant add nucleotide

Question 11

Phosphoramidite procedure

Answer 11

Procedure of oligonucleotide synthesis

DNA chain built in 3’ to 5’ direction

Coupling efficiency determines yield and length of oligonucleotide

Long dsDNA molecules can be produced by synthesising smaller overlapping oligonucleotides and fill gaps using PCR