Download Dr Ishtiaq Lecture at GC Faisalabad

Chemistry and Biology of DNA in the Age
of Personalized Medicines
Dr. Ishtiaq Ahmad Khan
Assistant Professor
Dr. Panjwani Center for Molecular Medicine and Drug Research
International Center for Chemical and Biological Sciences, University of
Karachi.
DNA in every organism
DNA – The molecule of Life
The building blocks of DNA are nucleotides.
each nucleotide composed of:
– a nitrogenous base
(adenine, thymine, guanine, cytosine
abbreviated as A, T, G, C)
– a 5-carbon sugar called deoxyribose
– a phosphate group (PO4)
Nucleotides
ester
linkage
Nitrogenous
base
(adenine)
Phosphate
group
5-carbon
Ribose sugar
DNA Structure
Nucleotides are connected to each other by
phosphodiester bonds to form a long chain
Double helical Structure of DNA
– 2 sugar-phosphate backbones
– nitrogenous bases toward the interior of the
molecule
– bases form hydrogen bonds with complementary
bases on the opposite sugar-phosphate backbone
• A pairs with T (2 H bonds)
• C pairs with G (3 H Bonds)
Noble Prizes in DNA Chemistry
Watson, Crick, and Wilkins (1962):
Discovery of structure of DNA
H. Gobind Khorana (1973)
Chemical synthesis of oligonucleotide
Berg, Gilbert, and Sanger (1980):
The determination of base sequences in nucleic acids
Mullis and Smith (1993):
Contributions to the developments of methods within
DNA-based chemistry. Invention of PCR
Sequencing the DNA
In the 1970’s, Sanger’s group discovered a
method of 'reading' the linear DNA sequence
using special nucleotide bases called chain
terminators or di-deoxy nucleotides.
This method is still
in use today.
Frederick Sanger
Died on November 19, 2013
Dideoxy Nucleotide
Next Generation DNA Sequencing
Massively parallel sequencing, Shotgun Genome Sequencing:
for sequencing the whole genome and whole exome
Complete genome
copies
Next Generation DNA Sequencing
Fragmented genome
chunks
NOT REALLY DONE BY DUCK HUNTERS !
Hydroshearing, sonication, enzymatic shearing
Major NGS Platforms
• 454 FLX Genome Sequencer (Roche)
Pyrosequencing method
• Hiseq2000, Solexa Genome Analyzer (Illumina)
Sequencing by seynthesis
• SOLiD ( Applied Biosystems)
Sequencing by ligation chemistry
• Ion-torrent (Life technologies)
An integrated semiconductor device enabling non-optical genome sequencing
• Heliscope (Heliscope Biosciences)
Single molecule sequencing by synthesis
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Assembly of short reads
17 bp
ATTGTTCCCACAGACCG
CGGCGAAGCATTGTTCC
ACCGTGTTTTCCGACCG
AGCTCGATGCCGGCGAAG TTGTTCCCACAGACCGTG TTTCCGACCGAAATGGC
ATGCCGGCGAAGCATTGT
ACAGACCGTGTTTCCCGA
TAATGCGACCTCGATGCC
AAGCATTGTTCCCACAG
TGTTTTCCGACCGAAAT
TGCCGGCGAAGCCTTGT
CCGACCGAAATGGCTCC
66 bp
Consensus sequence:
TAATGCGACCTCGATGCCGGCGAAGCATTGTTCCCACAGACCGTGTTTTCCGACCGAAATGGCTCC
Overview of DNA to RNA to Protein
•
A gene is expressed in two steps
– Transcription: RNA synthesis
– Translation: Protein synthesis
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
RNA
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
RNA
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleotides called codons.
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleotides called codons.
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleotides called codons.
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleotides called codons.
Amino acid 1 Amino acid 2
The Central Dogma: DNARNAProtein
DNA: A long double-stranded string of
nucleotides that encode for many
genes.
Gene
RNA: A single-stranded copy of one
gene.
Codon 1 Codon 2
Protein: Proteins are composed amino
acids. Amino acids are made from
triplets of nucleotides called codons.
Amino acid 1 Amino acid 2
Protein!
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
Amino Acids/Protein:
Met
GTG
CTG
TCT
CCT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
Amino Acids/Protein:
Met
Val
CTG
TCT
CCT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
Amino Acids/Protein:
Met
Val
Leu
TCT
CCT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
Amino Acids/Protein:
Met
Val
Leu
Ser
CCT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
Amino Acids/Protein:
Met
Val
Leu
Ser
CCT
Pro
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
Amino Acids/Protein:
Met
Val
Leu
Ser
CCT
Pro
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
Amino Acids/Protein:
Met
Val
Leu
Ser
Pro
DNA:
ATG
GTG
CTG
TCT
ACT
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
Amino Acids/Protein:
Met
Val
Leu
Ser
Pro
DNA:
ATG
GTG
CTG
TCT
ACT
Amino Acids/Protein:
Met
Val
Leu
Ser
Thr
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
Amino Acids/Protein:
Met
Val
Leu
Ser
Pro
Words:
Tom
and
Sam
are
bad
DNA:
ATG
GTG
CTG
TCT
ACT
Amino Acids/Protein:
Met
Val
Leu
Ser
Thr
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
Amino Acids/Protein:
Met
Val
Leu
Ser
Pro
Words:
Tom
and
Sam
are
bad
DNA:
ATG
GTG
CTG
TCT
ACT
Amino Acids/Protein:
Met
Val
Leu
Ser
Thr
Words:
Tom
and
Sam
are
sad
A small change in the gene sequence can result in a
very different protein
DNA:
ATG
GTG
CTG
TCT
CCT
Amino Acids/Protein:
Met
Val
Leu
Ser
Pro
Words:
Tom
and
Sam
are
bad
DNA:
ATG
GTG
CTG
TCT
ACT
Amino Acids/Protein:
Met
Val
Leu
Ser
Thr
Words:
Tom
and
Sam
are
sad
Changes in DNA are called variations or mutations
Variations in the DNA (genotype) can cause observable changes (phenotype) in individuals
Human Genome Research
• Human Genome Project in 2003
•Finishing the euchromatic sequence of the human genome.
•Nature 2004; 431 (7011): 931-945.
• Phase I HapMap project in 2005
•A haplotype map of the human genome.
•Nature 2005: 437(7063):1299-1320
• Encyclopedia of DNA Elements (ENCODE) project in 2007
•Identification and analysis of functional elements in 1%
•of the human genome by the ENCODE pilot project.
•Nature 2007; 447(7146):799-816
• 1000 Genomes Project in 2008
•DNA sequences. A plan to capture human diversity in 1000 genomes.
•Science 2008; 319(5863):395
Genotypes and Human Disease
Do all humans have the same DNA?
• Single nucleotide polymorphisms or SNPs.
• We can associate SNPs with medical histories of
individuals and achieve statistically significant
correlations.
• Pharmacogenetics (PGx) is the science of how an
individual’s genotype affects their body’s response
to drugs.
Variations
in our
DNA
make us
UNIQUE!
Genomics in Drug Discovery
Personalized Medicines
Personalized medicine is the use of
information from a patient's genotype to:
• Initiate a preventative measure against the
development of a disease or condition,
or
• Select the most appropriate therapy for a
disease or condition that is particularly
suited to that patient.
Why does it matter?
In treating all people with a certain lung cancer without having a
genetic test , 10 % of people might respond.
In treating people who have a genetic test and are found to have
a tumour mutation , 85% of people treated for that mutation
might respond.
The best drug for a specific disease in a
particular person.
Is Personalized medicine only for sick people?
Definitely not. Because an individual's genome influences his or
her likelihood of developing (or not developing) a broad range of
medical conditions, personalized medicine focuses strongly on
wellness and disease prevention.
If a person's genomic information indicates a higher-thanaverage risk of developing diabetes or a particular form of
cancer, that person may choose a lifestyle, or sometimes be
prescribed medications.
Examples of SNPs
Linked to Drug Response
Case Study: Warfarin
• Most widely prescribed oral anticoagulant for preventing
thrombolytic events, despite its narrow therapeutic range.
• Problematic dosing due to patient’s diet, age, and other
medications.
•CYP2D9/VKORC1 genes analysis predicts its prescription.
(Mol Interv. 2006, 6(4): 223-277)
Case Study: Irinotecan
• Irinotecan is used as chemotherapeutic agent for the
treatment of metastatic colorectal cancer.
• It produces adverse drug reactions in some patients.
• genotyping assay is carried out to mutations in UGT1A1
gene for irinotecan prescribing.
(Personalized Medicine 2006, 3(4): 415-419)
Case Study: Abacavir
• Abacavir is a nucleoside analog reverse transcriptase
inhibitor used to treat human immunodeficiency virus (HIV) .
• Its main side effect is hypersensitivity reaction (HSR).
• HSR is associated with ethnicity. A significantly increased risk
of abacavir-induced HSR in human leukocyte antigen (HLA)B*57:01-carrying patients.
Sci China Life Sci. 2013, 56(2):119-24
Interferon Therapy
• Standard approved therapy for Hepatitis C
• Differential response
• Interleukin 28B gene (IL28B) variants which
are associated with decreased HCV clearance
• Interleukin 28B gene (IL28B) variants are
considered as key players in deferential
therapeutic responses.
Malignant Melanoma 2007
Melanoma 2013
Targeting treatment to a specific variant in the Melanoma gene.
Structure of Vemurafenib (marketed as Zelboraf)
Beta-thalassemias
(β-thalassemias) are a group of inherited blood
disorders caused by problems in the production
of hemoglobin, the oxygen-carrying protein in red
blood cells.
Cause is the mutations in beta-chain, 200 SNPs and
several deletion
61
Hummmm……
62
Genomics
at
International Center for Chemical and
Biological
Current Research Projects
• Five human genome project
• Integrated analysis of HCV genome and Exome
of Hepatitis C patients to Study genetics of
differential response to interferon therapy
• Assessment of genetic factors for Ventricular
Septal Defect
• Genomics of Diabetic patients
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Memorable Moments
Red Fort (Lal Qila) Delhi
India
Education complex of Islamic era
at Qutab complex Delhi
Qutab Minar Delhi India