Sequences and their Properties

NucleotidesNucleotides

DNADNA A, T, G, CA, T, G, C

RNARNA A, U, G, CA, U, G, C

AnnealingAnnealing

Nucleic acids can base pair with their Nucleic acids can base pair with their reverse complement sequencereverse complement sequence

Two opposing forces affect Two opposing forces affect annealingannealing Hydrogen bonds favours Hydrogen bonds favours

annealingannealing Phosphate groups favours Phosphate groups favours

denaturationdenaturation

Annealing-Melting Point (Tm)Annealing-Melting Point (Tm) The Tm is the temperature at The Tm is the temperature at

which 50% of the nucleic acid which 50% of the nucleic acid molecules are in a single stranded molecules are in a single stranded state (or double stranded)state (or double stranded)

The Tm is a function of:The Tm is a function of: Percentage G:CPercentage G:C Ionic composition of the environmentIonic composition of the environment The percentage of complementarityThe percentage of complementarity

Estimate of TmEstimate of Tm =2(#A:T) + 4(#G:C)=2(#A:T) + 4(#G:C) 4

5

Tm Vs percentage G:C

70 80 90 100

0

50

100

% D

oubl

e st

rand

ed

Temperature (C)

(38%) G+C(52%)

(58%)

(66%)

6

Tm Vs Conc. of Positive Ions

70 80 90 100

% D

oubl

e st

rand

ed

Temperature (C)

(0.1M NaCl)

(0.2M NaCl)

(0.5M NaCl)

0

50

100

7

Tm Vs percentage of Complementarity

70 80 90 100

% D

oubl

e st

rand

ed

Temperature (C)

(25%)

(50%)

(100%)

0

50

100

StringencyStringency

Percentage of complementarity Percentage of complementarity required to allow the formation of stable required to allow the formation of stable duplexesduplexes

The Tm influences the stringency The Tm influences the stringency conditions required to allow annealingconditions required to allow annealing A high stringency requires a high level of A high stringency requires a high level of

complementaritycomplementarity

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GATCCGGTTATTA vs GATCCGGTTATTA CTAGGCCAATAAT CTTGGACGATAAT

Parameters that Influence Parameters that Influence StringencyStringency

[salt] = High stringency [salt] = High stringency Temperature = High stringency Temperature = High stringency [salt] = ? [salt] = ? Temperature = ?Temperature = ?

9

4. Hybridization with 4. Hybridization with FreeFree Probe Probe

Wash

Detection: Detection: AutoradiographyAutoradiography

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Properties of the ProbeProperties of the ProbeComplementarity

Complete or partial?Complete; ideal; 100% complementarity

Partial continuous; acceptable100% complementarity

Partial discontinuous; more difficutPartial complementarity

Hybridization StringencyHybridization Stringency

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The ProbeThe Probe

Labelled DNA or RNA moleculeLabelled DNA or RNA molecule

Single strandedSingle stranded

Strand specific (sense specific)Strand specific (sense specific)

Double strandedDouble stranded

Strand non-specific (sense non specific)Strand non-specific (sense non specific)

Digoxygenin Labelled ProbeDigoxygenin Labelled Probe

Indiret detection

S S S S

MembraneTarget

D D D D D D D D Probe+ Dig

ENZ ENZ ENZ ENZ

Ab-Dig conjugatedPeroxidase

X ray film

Hybridization SignalsHybridization Signals

HybridizationHybridization SpecificSpecific Non specificNon specific

BackgroundBackground Binding of probe to membrane Binding of probe to membrane Binding of Ab to membraneBinding of Ab to membrane

Decoding the Genetic Decoding the Genetic InformationInformation

Information encoded in nucleotide Information encoded in nucleotide sequences is contained in discrete sequences is contained in discrete unitsunits The genesThe genes

The information contained in the The information contained in the genes is genes is transcribedtranscribed to generate the to generate the RNAs and then decoded (RNAs and then decoded (translatedtranslated) ) to generate the proteinsto generate the proteins

Protein Coding SequencesProtein Coding Sequences Protein containing sequences Protein containing sequences

contain ORFscontain ORFs Start – ATGStart – ATG Stop – TAG, TGA, TAAStop – TAG, TGA, TAA

Transcription - TranslationTranscription - Translation

Transcription: RNA pol

Translation: Ribosomes

NH3-M-T-R-S-W-G-L-I-S-I-COOH

ORFsORFs All double stranded sequences All double stranded sequences

necessarily have 6 reading framesnecessarily have 6 reading frames

How many ORFs does this sequence have?

ATGCCGATTAGA>TGCCGATTAGAG>GCCGATTAGAGA>

<CTGTCGGTAATT<TCTGTCGGTAAT<CTCTGTCGGTAA

5’-ATGGCGATTAGAGACAGCCATTAA-3’3’-TACTGCTAATCTCTGTCGGTAATT-5’

HomologuesHomologues

Gene sequences that possess a Gene sequences that possess a common ancestor common ancestor Homologues share a high level of Homologues share a high level of

identityidentity IdentityIdentity

Percentage of bases or amino acids that Percentage of bases or amino acids that are the same between different are the same between different sequencessequences

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Nucleotide HomologuesNucleotide Homologues

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77% identity

DNA sequences with greater 70% DNA sequences with greater 70% identityidentity Ex. A homologue of the human Ex. A homologue of the human

hemoglobin gene is found in soyahemoglobin gene is found in soya

G.G.T.G.A.G.G.G.T.A.T.C.A.T.C.C.C.A.T.C.T.G

G.G.T.C.A.G.G.A.T.A.T.G.A.T.T.C.C.A.T.C.A.C

* * * * * * * * * * * * * * * *

Protein homologuesProtein homologues

Protein sequences with greater Protein sequences with greater than 25% identitythan 25% identity Ex. A protein homologue of the Ex. A protein homologue of the

human hemoglobin is found in soya human hemoglobin is found in soya

23Percentage identity: 28%

G A R G G W LG.G.T.G.A.G.G.G.C.A.T.C.A.T.C.C.C.A.T.C.TG.G.T.C.A.G.G.A.C.A.T.G.A.T.T.C.C.A.T.C.A G T P M I W E

HomologuesHomologues

Orthologues :Orthologues : Homologues found in different Homologues found in different

organismorganismss which have a common which have a common ancestor ancestor

Duplication followed by speciationDuplication followed by speciation

Paralogues : Paralogues : Homologues found within the same Homologues found within the same

speciesspecies Duplication prior to speciationDuplication prior to speciation

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MutationsMutations

Point MutationsPoint Mutations Missense - Neutral Synonymous/Silent :Missense - Neutral Synonymous/Silent :

Base change that does Base change that does NOTNOT change the amino acid change the amino acid codedcoded

Ex. AGG → CGG both ArgEx. AGG → CGG both Arg Missense - Non-Synonymous - Conserved:Missense - Non-Synonymous - Conserved:

Base change results in a different but similar amino Base change results in a different but similar amino acidacid

Same charge and shapeSame charge and shape Ex. AAA → AGA Lys to Arg both basic amino acidsEx. AAA → AGA Lys to Arg both basic amino acids

Point MutationsPoint Mutations Missense - Non-Synonymous-Semi conserved:Missense - Non-Synonymous-Semi conserved:

Base change resulting in a different but similar Base change resulting in a different but similar amino acidamino acid

Same shape but different chargeSame shape but different charge Ex. CGC → CUC Arg (Polar) to Leu (Non-polar)Ex. CGC → CUC Arg (Polar) to Leu (Non-polar)

Missense - Non-Synonymous - Non conservedMissense - Non-Synonymous - Non conserved Base change resulting in totally different amino Base change resulting in totally different amino

acidsacids Different shape different chargeDifferent shape different charge

Point MutationsPoint Mutations Nonsense point mutation:Nonsense point mutation:

Base change resulting in the creation of a Base change resulting in the creation of a premature stop codon within the ORFpremature stop codon within the ORF

Causes premature translation terminationCauses premature translation termination Truncated proteinTruncated protein

Indel – Insertion or deletion of a single base Indel – Insertion or deletion of a single base within the ORFwithin the ORF Changes reading frameChanges reading frame Changes protein sequenceChanges protein sequence May cause premature terminationMay cause premature termination