course: recombinant dna biology course code: mslsc2003c04 · course: recombinant dna biology course...
TRANSCRIPT
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
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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
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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
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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
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• Each ddNTP linked to a
– fluorescent molecule
– all fragments have a particular color
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• 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
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• The DNA sequence is read by
• determining the sequence of colors in peaks
• information is fed directly to a computer
• which determines the sequence
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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
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deoxyadenosine alpha-thio triphosphate (dATPαS) is used in mix as not recognized by luciferase
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adenosine 5´ phosphosulfate
visible light in amounts that are proportional to the amount of ATP
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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.
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• 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
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454 sequencing run
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Snapshots of 454 sequencing run
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• 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
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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
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Overlapping Alignment of sequencing
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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
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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
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PPI identification methods
• Yeast Two-Hybrid System
• Co-immunoprecipitation
• In-silico tools like BIND, MINT, IntAct
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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
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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
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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
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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
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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/
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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.
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