course: recombinant dna biology course code: mslsc2003c04 · course: recombinant dna biology course...

E-Learning Course: Recombinant DNA Biology

Course Code: MSLSC2003C04Course Coordinator: Dr. Tara Kashav

Topic: DNA Sequencing and macromolecular

interactionsNote: All the material is compiled/modified from

textbooks/freely available e-resources just meant for learning purpose of students

DNA sequencing

• Sanger sequencing

– Enzymatic method

– More popular

• Maxam and Gilbert

– Chemical method

– Suitable for short oligonucleotides

Dr. Tara Kashav, CUSB 2

Sanger and Maxam-Gilbert sequencing

• General principle:

– To reduce the DNA to four sets of labeled fragments

• The reaction producing each set is base-specific

– Lengths of the fragments correspond to positions in the DNA sequence where a certain base occurs


Sanger method or dideoxy method

• Uses dideoxynucleoside triphosphate (ddNTP) analogs

– to interrupt DNA synthesis

• When a ddNTP is inserted in place of a dNTP

– strand elongation is halted after the analog is added

– lacks the 3’-OH group needed for the next step


Automating DNA-sequencing

• Each ddNTP used in the Sanger method linked to a

– fluorescent molecule

– all the fragments terminating in that nucleotide a particular color


• Each ddNTP linked to a

– fluorescent molecule

– all fragments have a particular color


• All four labeled ddNTPs are added to

• single electrophoretic gel contained in a capillary tube

• colored DNA fragments are then separated by size

• color associated with each peak is detected using a laser beam


• The DNA sequence is read by

• determining the sequence of colors in peaks

• information is fed directly to a computer

• which determines the sequence


Pyrosequencing

• Sequencing by synthesis

• PCR template is hybridised to an oligonucleotide

– Incubated with DNA pol, ATP sulphurylase, luciferase and apyrase

• During the reaction

– On addition of first dNTP, release pyrophosphate (PPi)

– The ATP sulphurylase converts the PPi to ATP

– ATP drives luciferase-mediated conversion of luciferin to oxyluciferin to generate light –Signal recorded !

– Apyrase degrades the resulting component dNTPs and ATP


deoxyadenosine alpha-thio triphosphate (dATPαS) is used in mix as not recognized by luciferase


adenosine 5´ phosphosulfate

visible light in amounts that are proportional to the amount of ATP


Next-generation sequencing (NGS)

• Two widely utilized next-generation sequencers use

• 454 sequencer

– Uses a strategy called pyrosequencing

– Addition of nucleotides is detected with flashes of light

• Illumina sequencer

– Uses reversible terminator sequencing

• Typical flow cell used for NGS

– Millions of DNA fragments can be sequenced simultaneously in each of eight channels.


• four dNTPs are pulsed onto reacting surface one at a time, in a repeating sequence

• Nucleotide solution is retained on surface just long enough for DNA polymerase to add that nucleotide to any cluster where it is complementary to next template base in sequence

• Excess nucleotide is destroyed quickly with apyrase before the next nucleotide pulse

• When a specific nucleotide is successfully added to strands of a cluster, pyrophosphate is released as a byproduct


454 sequencing run

Snapshots of 454 sequencing run


• Each segment of DNA is attached to a tiny DNA capture bead, then amplified on the bead by PCR

• Each bead is immersed in an emulsion and placed in a tiny (29 µm) well on a picotiter plate

• Reaction of luciferin and ATP with luciferase produces light flashes when a nt is added to a particular well


Snapshots of 454 sequencing run

• Circles represent same cluster over multiple cycles

• In this case, reading the top (or bottom) circle from left to right across each row gives the sequence for that cluster.

Next-generation reversible terminator sequencing


Overlapping Alignment of sequencing


Seq from one strand (5’-3’)- solid arrowsSeq from other strand (3’-5’) - dashed lines

Zoomed in sequences shown at bottom

genomic base-pair positions relative to an arbitrarily defined “0.”

SNP statistics report

Techniques to study some crosstalk….

• DNA-PROTEIN INTERACTIONS

• PROTEIN-PROTEIN INTERACTIONS


Protein-Protein Interactions (PPI)

• PPI occurs when two or more proteins bind together

• Protein control/mediate many biological activities

• Information about PPIs helps in

– understanding of diseases

– provide basis for new therapeutic approaches

• Application:

– Analysis of metabolic and signal transduction to find out disease pattern

– Pharmacogenetics research to study drug transportes, drug receptors, and drug targets


PPI identification methods

• Yeast Two-Hybrid System

• Co-immunoprecipitation

• In-silico tools like BIND, MINT, IntAct


Yeast Two-Hybrid (Y2H) System

• Testing for physical interaction between two proteins

• Based upon properties of yeast GAL4 protein, that consists of separate domains for DNA-binding domain (DBD) and transcriptional activation domain (TAD)

• Plasmid encoding two hybrid proteins are constructed and introduced into yeast

– One consists of GAL4 DBD fused to protein X

– Other consists of GAL4 TAD fused to protein Y are

• Interactions between X and Y leads to transcriptional activation of reporter gene containing a binding site for GAL4



Yeast Two-Hybrid (Y2H) System

Co-immunoprecipitation

• Uses target protein-specific antibodies to indirectly capture proteins that are bound to specific target

• These protein complexes can then be analysed to

– Identify new binding partners

– Binding affinities

– Kinetics of binding

– Function of the target protein



Co-immunoprecipitation

In silico toolsBIND

• http://bind.ca

• A free, open source database for achieving and exchanging molecular assembly information

• The database contains:

– Interactions

– Molecular Complexes

– Pathways


http://bind.ca/

BIND

• BIND interaction viewer java showing how molecules can be connected in the databse from molecular complex to small molecule

– Yellow: Protein

– Purple: Small molecule

– White: molecular complex

• Ex: cell signalling in T-cell


Other Database

• DIP (Database of Interacting Proteins)

• https://dip.doe-mbi.ucla.edu/dip/Main.cgi

• MINT

• https://mint.bio.uniroma2.it/

• STRING

• https://string-db.org/

• Human Protein Interaction Database

• http://wilab.inha.ac.kr/hpid/


https://dip.doe-mbi.ucla.edu/dip/Main.cgi

https://mint.bio.uniroma2.it/

https://string-db.org/

http://wilab.inha.ac.kr/hpid/

Reading

• Lehninger’s Biochemistry book

• Primrose, S. B., & Twyman, R. (2009). Principles of gene manipulation and genomics. Wiley. com.

• Brown, T. (2010). Gene cloning and DNA analysis: an introduction. John Wiley & Sons.


course: recombinant dna biology course code: mslsc2003c04 · course: recombinant dna biology course...

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