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Topic List Notes
14. Meaning of Genetic Mutations and Polymorphisms and
Methods of their Analysis (RFLP, Allele-specific PCR, DNA
Sequencing, Primer Extension)
Genetic Mutation: change in DNA sequence that is not corrected, results in
permanent change. Occurs in <1% of population
Polymorphism: change in phenotype due to a change in DNA sequence. More than
one allele occupies that gene’s locus. Occurs in >1% of population
Sanger Sequencing: determines the order of nucleotides within DNA
Reaction Mixture: ssDNA template, primer, DNA polymerase, 4 dNTPs, add 1
ddNTP to each tube
Compares the length of ssDNA product to the original strand —> can find exact
position of base
4 Different reactions to determine the position of 4 bases via Gel electrophoresis
Primer extension: Primer ends NEXT to the site of mutation
Template strand is mRNA, primer anneals to its 3’ end and extends via reverse
transcriptase
cDNA is produced as a product
Determines the start site of transcription
Reaction Mixture: Primer, 4ddNTPs labeled with diff flourescent dyes
The primer is complementary to a certain region near the mutation point (if the
PCR product is thymine —> chain terminates at adenine)
Topic List Notes 1
, The product has no length difference, but has a color difference, showing which
base is there
Restriction Fragment Length Polymorphism (RFLP)
Uses a specific Restriction Endonuclease Type 2 which only cleaves one specific
DNA strand
So if there is a mutation, then cleavage won’t happen
This converts the sequence difference into length differences
Allele Specific PCR: Primer ends ON site of mutation
Uses a polymerase WITHOUT 3’ exonuclease activity so that elongation is stopped
at mutation, creating length differences
Uses primers that are a perfect match to the ssDNA sequence
If the primer is not a perfect match, due to an SNP, then elongation is stopped
The mutated allele won’t make any PCR product!
Each allele needs to reactions: In a C/T polymorphism you need one reaction for C
and one for T
II. RNA
Topic List Notes 2
, 15. Structure and Function of RNA polymerase of E.coli;
initiation of transcription in prokaryotes; the prokaryotic
transcription unit
Transcription = DNA segment is copied into RNA (mRNA), based on complimentary
base pairing
occurs in cytoplasm — prokaryotes don’t have a nucleus!!
performed by the RNA polymerase
DNA contains genes. A gene is a nucleotide sequence that codes for an RNA
molecule. This sequence begins with a promoter and ends with a terminator. RNA
polymerase binds to the promoter.
**Transcription is the first step towards gene expression (translating genotype —>
phenotype)
(Phases of Gene Expression)
Transcription to RNA —> RNA modification —> Translation into protein —>
Post-translational modifications
Major Differences to DNA replication:
1) Uracil instead of Thymine (A → U)
2) The Pentose Sugar is Ribose instead of deoxyribose
3) RNA synthesis starts with NO PRIMER (this is an explanation for why RNA
evolved before DNA in early life)
The first nucleotide added is usually ATP
4) There is no proofreading in RNA polymerase (and mistakes cannot be
inherited as the translated protein can just be degraded if its faulty)
2 Strands:
Template strand (Sense strand) (used to synthesize a new RNA strand)
The new RNA strand grows in 5’ → 3’ direction
Coding strand (Anti-sense strand)
Copying: 3’→ 5’ direction
Synthesis (growing): 5’→3’ direction
Topic List Notes 3
, *Some genes are encoded by one of the strands, while other are by the other strand!
Structure and Function of RNA polymerase
RNA polymerase substrates: ribonucleoside triphosphates (ATP, GTP, CTP,
TTP)
(Reminder, the DNA substrates are dATP, dGTP, dCTP, dTTP)
RNA Polymerase Composed of 5 subunits (Core Enzyme):
Small: α and α
-assembly of complex, interaction with the promoter, interactions with
regulatory factors
Large: β and β’
-together they form the catalytic center for RNA synthesis
-β’ is biggest (160kDa)
Stabilizer: σ subunit
Topic List Notes 4
Topic List Notes
14. Meaning of Genetic Mutations and Polymorphisms and
Methods of their Analysis (RFLP, Allele-specific PCR, DNA
Sequencing, Primer Extension)
Genetic Mutation: change in DNA sequence that is not corrected, results in
permanent change. Occurs in <1% of population
Polymorphism: change in phenotype due to a change in DNA sequence. More than
one allele occupies that gene’s locus. Occurs in >1% of population
Sanger Sequencing: determines the order of nucleotides within DNA
Reaction Mixture: ssDNA template, primer, DNA polymerase, 4 dNTPs, add 1
ddNTP to each tube
Compares the length of ssDNA product to the original strand —> can find exact
position of base
4 Different reactions to determine the position of 4 bases via Gel electrophoresis
Primer extension: Primer ends NEXT to the site of mutation
Template strand is mRNA, primer anneals to its 3’ end and extends via reverse
transcriptase
cDNA is produced as a product
Determines the start site of transcription
Reaction Mixture: Primer, 4ddNTPs labeled with diff flourescent dyes
The primer is complementary to a certain region near the mutation point (if the
PCR product is thymine —> chain terminates at adenine)
Topic List Notes 1
, The product has no length difference, but has a color difference, showing which
base is there
Restriction Fragment Length Polymorphism (RFLP)
Uses a specific Restriction Endonuclease Type 2 which only cleaves one specific
DNA strand
So if there is a mutation, then cleavage won’t happen
This converts the sequence difference into length differences
Allele Specific PCR: Primer ends ON site of mutation
Uses a polymerase WITHOUT 3’ exonuclease activity so that elongation is stopped
at mutation, creating length differences
Uses primers that are a perfect match to the ssDNA sequence
If the primer is not a perfect match, due to an SNP, then elongation is stopped
The mutated allele won’t make any PCR product!
Each allele needs to reactions: In a C/T polymorphism you need one reaction for C
and one for T
II. RNA
Topic List Notes 2
, 15. Structure and Function of RNA polymerase of E.coli;
initiation of transcription in prokaryotes; the prokaryotic
transcription unit
Transcription = DNA segment is copied into RNA (mRNA), based on complimentary
base pairing
occurs in cytoplasm — prokaryotes don’t have a nucleus!!
performed by the RNA polymerase
DNA contains genes. A gene is a nucleotide sequence that codes for an RNA
molecule. This sequence begins with a promoter and ends with a terminator. RNA
polymerase binds to the promoter.
**Transcription is the first step towards gene expression (translating genotype —>
phenotype)
(Phases of Gene Expression)
Transcription to RNA —> RNA modification —> Translation into protein —>
Post-translational modifications
Major Differences to DNA replication:
1) Uracil instead of Thymine (A → U)
2) The Pentose Sugar is Ribose instead of deoxyribose
3) RNA synthesis starts with NO PRIMER (this is an explanation for why RNA
evolved before DNA in early life)
The first nucleotide added is usually ATP
4) There is no proofreading in RNA polymerase (and mistakes cannot be
inherited as the translated protein can just be degraded if its faulty)
2 Strands:
Template strand (Sense strand) (used to synthesize a new RNA strand)
The new RNA strand grows in 5’ → 3’ direction
Coding strand (Anti-sense strand)
Copying: 3’→ 5’ direction
Synthesis (growing): 5’→3’ direction
Topic List Notes 3
, *Some genes are encoded by one of the strands, while other are by the other strand!
Structure and Function of RNA polymerase
RNA polymerase substrates: ribonucleoside triphosphates (ATP, GTP, CTP,
TTP)
(Reminder, the DNA substrates are dATP, dGTP, dCTP, dTTP)
RNA Polymerase Composed of 5 subunits (Core Enzyme):
Small: α and α
-assembly of complex, interaction with the promoter, interactions with
regulatory factors
Large: β and β’
-together they form the catalytic center for RNA synthesis
-β’ is biggest (160kDa)
Stabilizer: σ subunit
Topic List Notes 4
