why they want to make a map of the human genome · 2020-01-22 · maxam-gilbert sequencing is...

The project of mapping Human Genome

•Why they want to make a map of the human genome ?????

The project of mapping Human Genome

• The objective of sequencing human genome:

1. To understand how genes work together to direct the growth, development and maintenance of an entire organism.

2. By knowing the whole genome sequence it will help to study the parts of the genome outside the genes. This includes the long sequences of nonsense (junk) DNA that has no clear functions.

3. To learn about other important parts of the genome, such as the regulatory regions that help control when genes are turned on and off.

4. To draw accurate map for the chromosomal locations of genes responsible for genetic diseases.

Already, about 1400 genes are identified for human genetic diseases as a result of human genome mapping.

5. By comparing human genome map with other species maps, it will be possible to understand the

process of evolution.

The plan

The project of human genome sequencing began on 1990 and completed on 2003. The whole genome cannot be sequenced all at once because available methods of DNA sequencing can only work with short stretches of DNA at a time. Instead, the genome was broken into smaller pieces; approximately 150,000 base pairs in length. These pieces were cloned into plasmid vector before they were amplifies in bacterial culture.

• Using restriction enzymes, the pieces of human DNA were cut into small pieces and each gene was identified by specific probe before it was sequenced. Then the genes were reassembled in the proper order to obtain the sequence of the whole genome.

Data obtained from mapping human genome

1. The human genome sequence is almost exactly the same (99.9%) in all people.

2. There are approximately 23,000 genes in human beings being mapped, the same range as in mice and roundworms. Before this mapping process, the human genome was estimated to contain about

80-140 thousands genes, based on comparison with the size of bacteria in which the actual gene mapping already obtained.

3. The human genome contains 3.2 billion nucleotide base pairs .The average gene consists of 3,000 base pairs, but sizes vary greatly, with the largest known human gene has 2.4 million base pairs which is responsible for expressing the dystrophin protein.

4. Functions are still unknown for more than 50% of

discovered genes.

5. Genes appear to be concentrated in random areas

along the genome, with extended regions of

non-coding DNA in between.

6. Particular gene sequences have been found to be associated with various diseases, including breast cancer, muscle disease, deafness, and blindness.

7. Pieces of up to 30,000 C and G bases repeating over and over often occur adjacent to gene-rich areas, forming a barrier between the genes and the junk DNA. These C-G rich segments are believed to help in the regulation of gene activity.

8.Chromosome 1 (the largest human chromosome) has the most number of genes (3,168), and Y chromosome has the fewest number of genes (344).

Comparison of human genome with other organisms

1 . Unlike the random distribution of gene‐rich areas in human's, many other organisms'

genomes are more uniform, with genes evenly spaced throughout.

2. Humans have on average three times as many kinds of proteins as the fly or worm because of mRNA transcript alternative splicing and chemical modifications to the proteins. This process can yield different protein products from the same gene.

3. Humans share similar protein families with worms, flies, and plants, but the number of gene family members are more expanded in humans, especially in proteins involved in development and immunity.

4. The human genome has a much greater portion (50%) of repeat sequences than the mustard weed plant (11%), the worm (7%), and the fly (3%).

5. Over 40% of predicted human proteins share similarity with fruit‐fly or worm proteins

6. As a conclusion from this mapping sequence of human genome, it is believed that the quality of protein types produced by the genome is more important in providing the overall human phenotype than the number of genes that express these proteins.

DNA sequencing

Why sequence DNA?

• All genes available for an organism to use -- a

very important tool for biologists

• Not just sequence of genes, but also positioning

of genes and sequences of regulatory regions

• New recombinant DNA constructs must be

sequenced to verify construction or positions of

mutations

Sequencing Methods

• Maxam/Gilbert chemical sequencing

• Sanger chain termination sequencing

• Pyrosequencing

• Bisulfite Sequencing

• Array sequencing

Maxam-Gilbert sequencing is performed by chain

breakage at specific nucleotides.

DMS

G

G G

G

FA

G A

G G

A G

A A

H

C T

T C

T C

C T

H+S

C C

C

C

Maxam-Gilbert Sequencing

A. Maxam-Gilbert chemical cleavage method: DNA is labelled and

then chemically cleaved in a sequence-dependent manner. This

method is not easily scaled and is rather tedious

Sequencing gels are read from bottom to top (5′ to 3′).

G G+A T+C C

3′

A

A

G

C

A

A

C

G

T

G

C

A

G

5′

Longer fragments

Shortest fragments

G

A

Maxam-Gilbert Sequencing

Chain terminates

at ddG

Chain Termination (Sanger) Sequencing

Sanger dideoxy (primer extension/chain-termination)

method: most popular protocol for sequencing, very

adaptable, scalable to large sequencing projects

The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs.

Template area to be sequenced

-3′ OH TCGACGGGC…

5′OP-

Primer

Template


• A sequencing reaction mix includes labeled primer and template.

• Dideoxynucleotides are added separately to each of the four tubes.

ddATP + ddA four dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddC four dNTPs dAdGdCdTdGddC

dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC

ddGTP + dAddG four dNTPs dAdGdCdTddG

dAdGdCdTdGdCdCdCddG

ddTTP + dAdGdCddT four dNTPs dAdGdCdTdGdCdCdCdG

A

C

G

T



• With addition of enzyme (DNA polymerase), the primer is extended until a ddNTP is encountered.

• The chain will end with the incorporation of the ddNTP.

• With the proper dNTP:ddNTP ratio, the chain will terminate throughout the length of the template.

• All terminated chains will end in the ddNTP added to that reaction.


• The collection of fragments is a sequencing ladder.

• The resulting terminated chains are resolved by electrophoresis.

• Fragments from each of the four tubes are placed in four separate gel lanes.

Sequencing gels are read from bottom to top (5′ to 3′).

G A T C

3′

G

G

T

A

A

A

T

C

A

T

G

5′

Longer fragments

Shorter fragments ddG

ddG



• A modified DNA replication reaction.

• Growing chains are terminated by dideoxynucleotides.

for dideoxy sequencing you need:

1) Single stranded DNA template

2) A primer for DNA synthesis

3) DNA polymerase

4) Deoxynucleoside triphosphates and

dideoxynucleotide triphosphates

Primers for DNA sequencing

• Oligonucleotide primers can be synthesized by phosphoramidite chemistry--usually designed manually and then purchased

• Sequence of the oligo must be complimentary to DNA flanking sequenced region

• Oligos are usually 15-30 nucleotides in length

DNA templates for sequencing:

• Single stranded DNA isolated from recombinant

M13 bacteriophage containing DNA of interest

• Double-stranded DNA that has been denatured

• Non-denatured double stranded DNA (cycle

sequencing)

Reagents for sequencing:

DNA polymerases

• Should be highly processive, and incorporate ddNTPs efficiently

• Should lack exonuclease activity

• Thermostability required for “cycle sequencing”

Single stranded DNA 5’ 3’

5’ 3’

Sanger dideoxy sequencing--basic method

a) Anneal the primer

Sanger dideoxy sequencing: basic method

b) Extend the primer

with DNA polymerase

in the presence of all

four dNTPs, with a

limited amount of a

dideoxy NTP (ddNTP)

5’

3’

Direction of

DNA

polymerase

travel

Sanger dideoxy sequencing: basic method

5’ 3’

5’ 3’

T T T T

ddA

ddA

ddA

ddA

ddATP in the reaction: anywhere there’s a T in the template strand, occasionally a ddA will be added to the growing strand

Primer Walking

How to visualize DNA fragments?

• Radioactivity

– Radiolabeled primers (kinase with 32P)

– Radiolabelled dNTPs (gamma 35S or 32P)

• Fluorescence

– ddNTPs chemically synthesized to contain fluoresces

– Each ddNTP fluoresces at a different wavelength allowing identification

Analysis of sequencing products:

Polyacrylamide gel electrophoresis--good

resolution of fragments differing by a single

dNTP

– Slab gels: as previously described

– Capillary gels: require only a tiny amount of

sample to be loaded, run much faster than

slab gels, best for high throughput

sequencing.

DNA sequencing gels: old school

Analyze sequencing

products by gel

electrophoresis,

autoradiography

Different ddNTP used in

separate reactions

Radioactively labelled primer or dNTP in sequencing reaction

cycle sequencing: denaturation

occurs during temperature cycles

94°C:DNA denatures

45°C: primer anneals

60-72°C: thermostable DNA

pol extends primer

Repeat 25-35 times

Advantages: don’t need a lot of

template DNA

Disadvantages: DNA pol may

incorporate ddNTPs poorly

An automated sequencer

The output

Current trends in sequencing:

It is rare for labs to do their own sequencing:

--costly, perishable reagents

--time consuming

--success rate varies

Instead most labs send out for sequencing:

--You prepare the DNA (usually plasmid, M13, or PCR product),

supply the primer, company or university sequencing center does the

rest

--The sequence is recorded by an automated sequencer as an

“electropherogram”

Sequencing large pieces of DNA:

the “shotgun” method

• Break DNA into small pieces (typically sizes of around 1000 base pairs is preferable)

• Clone pieces of DNA into M13

• Sequence enough M13 clones to ensure complete coverage (eg. sequencing a 3 million base pair genome would require 5x to 10x 3 million base pairs to have a reliable representation of the genome)

• Assemble genome through overlap analysis using computer algorithms, also “polish” sequences using mapping information from individual clones, characterized genes, and genetic markers

• This process is assisted by robotics

~160 kbp

~1 kbp

Assemble sequences by matching overlaps

BAC sequence

BAC overlaps give genome sequence

BREAK UP THE GENOME, PUT IT BACK TOGETHER

Sequence by DNA polymerase -dependent chain extension, one base at a time in the presence of a reporter (luciferase) Luciferase is an enzyme that will emit a photon of light in response to the pyrophosphate (PPi) released upon nucleotide addition by DNA polymerase Flashes of light and their intensity are recorded

Height of peak indicates the number of dNTPs added

This sequence: TTTGGGGTTGCAGTT

Cycle Sequencing

• Cycle sequencing is chain termination sequencing performed in a thermal cycler.

• Cycle sequencing requires a heat-stable DNA polymerase.

Fluorescent Dyes

• Fluorescent dyes are multicyclic molecules that absorb and emit fluorescent light at specific wavelengths.

• Examples are fluorescein and rhodamine derivatives.

• For sequencing applications, these molecules can be covalently attached to nucleotides.

Fluorescent Dyes

• In dye primer sequencing, the primer contains fluorescent dye–conjugated nucleotides, labeling the sequencing ladder at the 5′ ends of the chains.

• In dye terminator sequencing, the fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains.

ddA

ddA

AC

GT

The fragments are distinguished

by size and “color.”

Dye Terminator Sequencing

• A distinct dye or “color” is used for each of the four ddNTP.

• Since the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube.

A

T

G

T

Capillary

G T C T G A

Slab gel

GA TC G A T C


The DNA ladder is resolved in one gel lane or in a capillary.

• The DNA ladder is read on an electropherogram.

Capillary Slab gel

5′ AGTCTG

Electropherogram


5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG

T/T T/A A/A

Automated Sequencing

• Dye primer or dye terminator sequencing on capillary instruments.

• Sequence analysis software provides analyzed sequence in text and electropherogram form.

• Peak patterns reflect mutations or sequence changes.

Alternative Sequencing Methods: Pyrosequencing

• Pyrosequencing is based on the generation of light signal through release of pyrophosphate (PPi) on nucleotide addition.

– DNAn + dNTP DNAn+1 + PPi

• PPi is used to generate ATP from adenosine phosphosulfate (APS).

– APS + PPi ATP

• ATP and luciferase generate light by conversion of luciferin to oxyluciferin.

DNA sequence: A T C A GG CC T

Nucleotide added : A T C A G C T

Alternative Sequencing Methods: Pyrosequencing

• Each nucleotide is added in turn.

• Only one of four will generate a light signal.

• The remaining nucleotides are removed enzymatically.

• The light signal is recorded on a pyrogram.

Alternative Sequencing Methods: Bisulfite Sequencing

• Bisulfite sequencing is used to detect methylation in DNA.

• Bisulfite deaminates cytosine, making uracil.

• Methylated cytosine is not changed by bisulfite treatment.

• The bisulfite-treated template is then sequenced.

The sequence of treated and untreated templates is compared.

GTC

Methylated sequence: GTC Me

GGC Me

GATCTATC Me

GTGCA …

Treated sequence: Me

GGC Me

GATUTATC Me

GTGUA …

DNA Sequence:

(Untreated) reference: ...GTCGGCGATCTATCGTGCA…

Treated sequence: ...GTCGGCGATUTATCGTGUA…

This sequence indicates that these Cs are methylated.

Bisulfite Sequencing

DNA

RNA

protein

genome

“transcriptome”

“proteome”

(we have this)

(we want these)

DNA microarray -- immobilize many probes

(thousands) in an ordered array, hybridize (base

pair) with labelled mRNA or cDNA

• Generating an array of probes

• Identify open reading frames (orfs)

1) PCR each orf (several for each orf), attach (spot)

each PCR product to a solid support in a specific

order (pioneered by Pat Brown’s lab, Stanford)

2) Chemically synthesize orf-specific oligonucleotide

probes directly on microchip (Affymetrix)

A yeast array experiment vegetative sporulating

Isolate mRNA

Prepare fluorescently labeled cDNA with two different-colored fluors

hybridize read-out

Example microarray data

Green: mRNA

more abundant in

vegetative cells

Red: mRNA more

abundant in

sporulating cells

Yellow: equivalent

mRNA abundance in

vegetative and

sporulating cells

DNA Microarrays: An Introduction

Microarray Result: Much analysis to follow

Microarray Technology

The value of DNA microarrays for

studying gene expression

1) Study all transcripts at same time

1) Transcript abundance usually correlates with

level of gene expression--much gene control is at

level of transcription

2) Changes in transcription patterns often occur as

a response to changing environment--this can be

detected with a microarray

Summary

• Genetic information is stored in the order or sequence of nucleotides in DNA.

• Chain termination sequencing is the standard method for the determination of nucleotide sequence.

• Dideoxy-chain termination sequencing has been facilitated by the development of cycle sequencing and the use of fluorescent dye detection.

• Alternative methods are used for special applications, such as pyrosequencing (for resequencing and polymorphism detection) or bisulfite sequencing (to analyze methylated DNA).

END Part I

why they want to make a map of the human genome · 2020-01-22 · maxam-gilbert sequencing is...

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