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Sequences and their Properties
NucleotidesNucleotides
DNADNA A, T, G, CA, T, G, C
RNARNA A, U, G, CA, U, G, C
Writing sequencesWriting sequences
Written 5’-3’Written 5’-3’ ATGGGTAGCGGTCATGATACATGGGTAGCGGTCATGATAC
ComplementComplement TACCCATCGCCAGTACTATGTACCCATCGCCAGTACTATG
Reverse (inverse)Reverse (inverse) CATAGTACTGGCGATGGGTACATAGTACTGGCGATGGGTA
Reverse complementReverse complement GTATCATGACCGCTACCCATGTATCATGACCGCTACCCAT
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) 5
6
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%)
7
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
8
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 required Percentage of complementarity required to allow the formation of stable duplexesto allow the formation of stable duplexes
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
GATCCGGTTATTA vs GATCCGGTTATTA CTAGGCCAATAAT CTTGGACGATAAT
9
Parameters that Influence Parameters that Influence StringencyStringency
[salt] = High stringency [salt] = High stringency Temperature = High stringency Temperature = High stringency [salt] = ? [salt] = ? Temperature = ?Temperature = ?
10
11
Method: Transfer and Immobilization onto Solid Support
Filter paper - wick
GelWell
Membrane
Filter paper
Absorbant paperWeight
20X SSC Solution– 3M NaCl, 0.3M NaCit.
12
4. Hybridization with Free Probe
Wash
13
Detection: Autoradiography
14
Properties of the ProbeComplementarity
Complete or partial?
Complete; ideal; 100% complementarity
Partial continuous; acceptable100% complementarity
Partial discontinuous; more difficutPartial complementarity
15
Hybridization Stringency
The Probe
Labelled DNA or RNA molecule
Single stranded
Strand specific (sense specific)
Double stranded
Strand non-specific (sense non specific)
Indiret detection
Digoxygenin Labelled Probe
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 Signals
HybridizationSpecificNon specific
BackgroundBinding of probe to membrane Binding 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
23
Nucleotide HomologuesNucleotide Homologues
24
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 hemoglobi is found in soya human hemoglobi is found in soya
25Percentage 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
26
MutationsMutations
Types of Missense Point Types of Missense Point MutationsMutations Neutral Synonymous/Silent :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 Non-Synonymous - Conserved: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
Types of Missense Point Types of Missense Point MutationsMutations Non-Synonymous-Semi conserved: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)
Non-Synonymous - Non conservedNon-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