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The Human Genome
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Dr. Hasan Alhaddad Guest lecturer: Molecular Basis of Human Diseases
October 12th, 14th, 16th 2014 Room 244 (1 PM)
Lectures structure
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• Part I (Sunday Oct 12th): • The book of life (Matt Ridely’s analogy with
modifications). • Introduction to the technologies at the time.
• Part II (Tuesday Oct 14th): • Why sequencing genomes/the human genome? • Genome war (public and private projects). • Sequencing the genome. • Genome assembly. • Genome annotation.
• Part III (Thursday Oct 16th): • Genome outcome. • The Genomic era.
AIMS (part I)
• Learn the basics of the human genome and understand how to simplify the concept of a genome to the public.
• Understand the technologies of the time and how they affect the project.
• Understand the limitations of the technologies.
• Understand some the general motives for sequencing the human genome.
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What is the genome?
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The book of life The code to our existence
The instructions to make who we are The map to how we look, feel, think, and behave
The genome is ourselves in a chemical language
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The human book of life – an analogy
The human book of life – an analogy
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I have two books
The human book of life is called
THE GENOME
The human book of life – an analogy
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Can you define the genome?
What cells in your body have one copy of the genome and what cells have two?
Some details on the human book of life
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• Humans are diploid organisms - two books (genomes) in most of their cells.
• Each book (genome) is composed of 23 chapters (chromosomes).
• The total number of chapters (chromosomes) in a humans is 46.
• Approximately 3 billion letters in the book!
• Sex determination depends on chapter 23 (the sex chromosomes).
• XX ! female. • XY ! male.
Autosomal chromosomes
Sex chromosomes
The human book of life
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How did we learn about the number of chromosomes in the human genome?
Sex determination (mostly)
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X X X Y
What are the 23rd chapters of your books of life?
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Genome size How did we learn about the size of the human genome?
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Genome size
How did we learn about the size of the human genome?
More details about the book
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• The number of pages, word,
and letters differ in each
chapter (chromosome).
• Chapters (chromosomes)
are numbered based on
their size. (chr 1 is the
largest).
• The instructions/readable
sections (genes) are not
equally distributed over the
chromosomes.
• Many sections of the book
(genome) are not readable.
• Many sections of the book
are of repeated letter,
words, or sentences.
• The book is written in a
chemical language
composed of four letters
(A,T,G,C).
• Sentences are made of
words made of three
letters (AAC, ATG, etc.).
What is written and how to read it?
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ATGCCATCACAAATGCGGCTATGCCATGACAAATGCGGCTAATGCCATGACAAATGCGGCTAATGCCATGACAAATGCGGCTAATGCCATGACAAATGCGGCTACATGACAAATGCGGCTAATGCCATGACAAATGCGGCTAATCATGACAAATGCGGCTACATGACCATGACAAATGCGGCTACATGACAACAACAACAACAACAACAACAACAACAACATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT
Met-Pro-Ser-Met-Arg-Lue-Cys-His-Asp-Lys-Cys-Gly-stop
Bla bla bla bla bla
More bla bla bla bla
Make brown eyes and stop
The genome in a cell
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• The Wellcome collection in London. • The human genome printed using font
size (5)! • If we print the genome using font size
12 and stretch the letter, it would go ~ from Kuwait to Spain!
• A lot of information. How is it packaged in a 100 trillion tiny little human cells (1-100 um)?
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Genome packaging
The genome is packaged via the interaction of DNA with
proteins.
Lost?
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Are you lost with these analogy?
If yes, do not worry.
You will learn all the details during upcoming lectures "
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What was known at the time? A summary: • Humans have diploid genome (2n = 46 chromosomes) in
their somatic cells.
• Humans have haploid genome (n = 23 chromosomes) in their germ-line cells.
• Humans have 22 pairs of autosomal chromosomes and 1 pair of sex chromosomes (X and Y)
• Human genome contains heterochromatic regions (highly condensed – few active genes) and Euchromatic region (lightly condensed – many active genes).
• Human genome size is ~ 3 billion bp.
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Molecular technologies of the time
How were genes/genomes studied at the time?
We will go over some key technologies that are relevant to the human genome project.
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Molecular technologies of the time Polymerase Chain Reaction
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Polymerase chain reaction
• Polymerase Chain Reaction (PCR) allows the amplification (copying) of small amounts of DNA millions of copies.
• The method was developed by Kary Mullis (1983) and he was awarded the Nobel Prize for his invention.
• The process of PCR is similar to the process of DNA replication except it is done in tubes rather than living cells.
• It is considered in many cases the first step before any genetic analysis.
• Many methods and applications involve PCR.
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DNA replication and PCR • DNA replication in the cells involves making an identical
copy of the genome (DNA).
• PCR uses the same procedure but to generate millions of copies of a small section of the genome in a tube!
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Components
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What do we need to replicate (copy) DNA?
1. DNA template.
2. Building block of DNA (dNTPs).
3. DNA copier (an enzyme).
4. 3’OH (primer).
Components: (1) DNA template
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• The DNA sample you collect from a crime scene or the one under investigation is the DNA template.
G A
C T
A
T
T
A
G
C
G
C
A
T
C
G
C
G
G
C
T
A
A
T
T
A
G
C
A
T
C
G
A
T
C
G
5’
5’
3’
3’
C T T A C C T G G C A T A C T G T G 5’ 3’
G A A T G G A C C G T A T G A C A C 5’ 3’
Each strand serves as a template for copying.
Remember complementary base-pairing!
Components: (2) dNTP (building blocks)
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Do you remember? DNA is made of nucleotides!
Deoxyribonucleoside triphosphate (dNTP)
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Four dNTPs serve as the building blocks of DNA (dATP, dTTP, dGTP, dCTP)
Remember Nucleotides!
Components: (2) dNTP (building blocks)
H H
Deoxyribonucleoside triphosphate (dNTP)
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Why triphosphate?
For the energy required to for the phosphodiester bond
Components: (2) dNTP (building blocks)
+
H H
Components: (3) DNA copier (polymerase)
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• DNA polymerase is the DNA copier in the cell.
• Uses the dNTPs (DNA building blocks) to make a complementary strand to the template.
• Uses the available 3’-OH of a previous nucleotide and 5’phsphate from dNTP to form a phosphodiester bond.
• Each time DNA Pol finds the correct complementary dNTP and catalyzes the reaction linking the new nucleotide.
Remember DNA Pol needs 3’-OH
Primers are short piece of polynucleotide
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G
C T
A
T
T
A
G
C C T G G C A T A C T G T G
5’
5’
OH-3’
3’
In order for the DNA copying machine to work and add nucleotides,
a 3’-OH needs to be available to form a phosphodiester bond!
Components: (4) primer (3’ OH)
PCR Process
• Three steps are involved in PCR:
1. DNA template denaturation: separation of the two strands of DNA.
2. Primers annealing: small oligonucleotide attaches to each separated strand providing the 3’OH for DNA polymerase.
3. DNA polymerization (extension): DNA polymerase extends the primers on both strands and adds nucleotides.
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PCR Process
1. DNA denaturation 2. Primers annealing 3. DNA extension
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PCR cycles
1. DNA denaturation 2. Primers annealing 3. DNA extension
Tem
p (C
)
94 C
55-65 C
72 C
What happens if we repeat this cycle many times?
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5
21 22 23 24 25
Exponential growth in the number of copies generated. The number of copies you get at the end of your PCR will be
2#cycles (236 cycles = 68 billion copies)
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PCR – how many copies?
Threshhold
Not enough DNA template
No chemicals left in the reaction tube
# D
NA
copi
es
# cycles
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Problems!
There were some difficulties with this system:
1. Three water-baths with three different temperature.
2. DNA polymerase denatures at 94 C.
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Problems!
DNA denature (94 C)
Primer annealing (55-65 C)
Extension (72 C)
Adding DNA Pol
DNA Pol denatures
• The sample has to be transferred into multiple water baths to accommodate the needed temperature.
• DNA polymerase needs to be added in every cycle because DNA polymerase denatures at high temperature.
Improvement 1
• Using Thermus aquaticus (Taq) polymerase.
• Taq polymerase is heat stable and the cycles can take place without the polymerase being destroyed during the denaturation phase.
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Improvement 1 • Using Thermus aquaticus (Taq) polymerase.
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Improvement 2
Replacing old machine (water baths) with a thermocycler.
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Improvement 2 Replacing old machine (water baths) with a thermocycler.
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To consider
• Length and GC content of your primer.
• Compatibility of your forward and reverse primers.
• Primer’s sequences do not complement each other (primer dimer).
• Annealing temperature of both primers should be the same.
• Length of the target DNA piece ( the longer the target the longer the extension time).
• DNA polymerase, primers and other chemicals’ concentration should be precisely calculated.
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Problem with PCR
What if we do not know the sequence of DNA to design primers?
How can I get enough copies of DNA?
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Problem with PCR
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• Copying unknown DNA using a biological system (bacteria).
• The approach used is called “Molecular Cloning”.
• It makes use Endonucleases (restriction enzymes) and plasmids of bacteria to copy a specific unknown piece of DNA.
• The plasmid’s DNA can serve as a primer for PCR and DNA sequencing.
Why copy DNA?
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Why molecular cloning or PCR matters in genome studies?
DNA CANNOT be sequenced using one copy!
What is DNA sequencing?
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It is reading the letters of the book. It is reading the exact nucleotide sequence of the genome.
Sequencing methods available now!
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1. Maxam and Gilbert chemical degradation method (extinct).
2. Sanger sequencing (dideoxy or chain termination method).
3. Illumina sequencing.
4. SOLiD sequencing.
5. Pyrosequencing.
6. Ion Torrent method.
7. Single molecule sequencing.
Sanger sequencing (the great method)
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• Fredrick Sanger has developed a sequencing method and received a Noble prize for it.
• Sanger sequencing method is also called Chain Termination Method and Dideoxy sequencing method.
• Employs: • specific primers
• dNTPs
• ddNTPs
• DNA polymerase
• DNA template
DNA synthesis
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+
H H
DNA synthesis requires the availability of a 3’-OH and energy
H H
DNA synthesis
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Difference in OH location in sugar and consequences
DNA synthesis
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The absence of OH group on the 3’ carbon of the sugar blocks further addition of nucleotides
Sanger sequencing procedure
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ddGTP ddCTP ddATP ddTTP
DNA Template
Polymerase
Excess dNTPs
Primer
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A G T A T G A A T T C 3� 5�
3� 5�
5� G
5� G A
5� A G A
5� A G A T
5� A G T A T
A G T A T 5� C
C 5� A G C T A T
A G C T A T T 5� C
5� 5� G 5� G A 5� A G A 5� A G A T 5� A G T A T A G T A T 5� C C 5� A G C T A T A G C T A T T 5� C
Sanger sequencing procedure
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A T G C
+
3�
5�
• Analysis using high resolution polyacrylamide gel electrophoresis.
• Fragments are detected using radioactive markers and autoradiography.
ACTGGTCAATCGATCGTA
Sanger sequencing procedure
_
Sanger sequencing - Gel
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• Reading these gels for the human genome is a lot of work.
• We need a faster more efficient method.
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• Each dideoxy nucleotide is attached to a florescent marker.
• At the end of each cycle, a laser beam can detect the florescent marker and thus record the position of the nucleotide.
Sanger sequencing - Automated
Chromatogram - Automated
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