Genes, Genomes & Microarrays

8/17/2019 Genes, Genomes & Microarrays

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Genes, Genomes & Microarrays

5BBB0231: Gene Cloning & expression



Molecular approaches to neurobiology

Principle of forward/reverse genetics

The Central Dogma

genomes, genes, mRNA, proteins

Finding interesting developmental genes

microarray approaches

other methodologies

Technologies to determine gene function

Cloning genes

RT-PCR

in silico mining

In s itu hybridisation

Functional Interference

Electroporation

Transgenic animalsmRNA knockdown







Watson & Crick model of DNA

structure

1955



DNA structure

Doublestranded

Stabilised by

base pairs

(A-T; C-G)through

hydrogen

bonds

Antiparallel

5’

5’

3’

3’





A = T, two hydrogen bonds

G ≡ C, three hydrogen bonds

Watson-Crick base pairing



DNA structure Right handed helix

10 nucleotides per turn

1 turn = 3.4 nm

Helix diameter = 2 nm



Hydrogen bonds in base pairs are not

covalent, and can be disrupted by heat or

chemicals

Heat Cool

“DNA

Hybridisation”



Hydrogen bonds in base pairs are not

covalent, and can be disrupted by heat or

chemicals

AlkaliLow [Na+]

Cool



DNA packaged into chromosomes.

In humans 46 chromosomes (22

pairs and XX or XY).3.2 x 109 nucleotide pairs

1.8 metre DNA in every nucleus

(4 µM diameter).

If each nucleotide was 1 mmapart …



What is in the human genome?

Human = 1 Trillion Cells

Each Cell = 3.2 x109 bp @ 0.34nm/bp

= 1 metre DNA per cell

= 1 Trillion metres

= 1 billion Km

6.7 x to the Sun and back!!!!= approx 1 light hour of information



DNA is supercoiled. In eukaryotic cells,

extra coiling is required to allow

packaging into nucleus

Beads on a stringappearance of

nucleosomes

F t i t t



From genome to interactome



Primary Reasons for Sequencing the Human Genome

• Complete sequencing of all genes

• Determine intron/exon structure of all genes

• Reveal non-coding regulatory sequences

• Identify polymorphisms

• Develop methodology for sequencing other genomes

• Uncover the unexpected



Step 1: Make a genomic library from a DNA sample

Comparison of different DNA cloning vectors which could be used

-----------------------------------------------------------------------------------------

Vector Host Structure Insert size (kb)

-----------------------------------------------------------------------------------------

Plasmids E. coli Circular plasmid 1-10

Cosmids E. coli Circular plasmid 35–45

BAC E. coli Circular plasmid Up to 300

PAC E. coli Circular plasmid 100–300

YAC S. cerevisiae Linear chromosome 100–2000

-----------------------------------------------------------------------------------------

How to sequence big genomes



YACsBACs orPACs

… . .

G A T C C A T C T A A T A C A …

. .

sequence

Plasmidsubclones

Step 3: Sub-assembly and Sequence



A (BAC) View of the Human Genome Project



Genebuild: 37.1 (hg19)

Known genes: 22,286

Novel genes: 34Pseudogenes: 12,308RNA genes: 9,922Gene transcripts 142,707

Base Pairs: 3,272,480,987

Current State of the Human Genome



Centromeres are highlyrepetitive DNA sequencesthat are difficult tosequence using currenttechnology. Millions (possiblytens of millions) of basepairs long

Telomeres, are highly repetitive:(TTAGGG)n

Vary in length between species from afew hundred bp in ciliates to

thousands of bp in vertebrates

Sequencing is still incomplete !telomere

telomere

centromere



The National Center forBiotechnology Information

www.ncbi.nlm.nih.gov

Hold all genomes in a databaseknown as Genbank, along withsequences of known andhypothetical genes.

Database lets you examine:

DNA sequence

Mutations and diseases

associated with particular genes

Body tissues in which this gene isactivated

papers written about this gene.etc etc



Genome Browsersat

University of California, Santa Cruz (UCSC)http://genome.ucsc.edu

and ENSEMBLhttp://www.ensembl.org



What other organisms have been sequenced and why?

Study evolution and diversity among species

Disease organisms (bacteria, viruses, parasites)

Vector of disease (eg. mosquito)

Commercial (fish and crops)

Laboratory model organisms for developmental biology,disease and genetics



What are the comparative genome sizes of humans and other organisms being studied?

organism estimatedsize

estimatedgene number

average genedensity

chromosomenumber

Homo sapiens (human) 3.2 giga bases ~22,000 1 gene per

100,000 bases

46

Rattus norvegicus (rat) 2.75 giga bases ~22,000 1 gene per100,000 bases

42

Mus musculus (mouse) 2.5 giga bases ~22,000 1 gene per100,000 bases

40

Tetraodon nigroviridis (pufferfish) 380 mega bases ~20,000 1 gene per 14,000

bases

21

Drosophila melanogaster (fruit fly) 1.8 mega bases 13,600 1 gene per 9,000bases

8

Arabidopsis thaliana (plant) 1.25 mega bases 25,500 1 gene per 4000bases

10

Caenorhabditis elegans (roundworm) 97 mega bases 19,100 1 gene per 5000bases

12

Saccharomyces cerevisiae (yeast) 12 mega bases 6300 1 gene per 2000bases

32

Escherichia coli (bacterium) 4.7 mega bases 3200 1 gene per 1400bases

1

H. Influenzae (bacterium) 1.8 mega bases 1700 1 gene per 1000bases

1



A Revision of Basic Gene Structure

transcription start site

promoter

Transcription factors and RNA polymerase II assemble at the promoter

What are the classic sequence signals that identify a promoter?

Down-stream or 3’

Up-stream or 5’

exons

introns

CpG

Other control sequences which interact with the promoter maybe found elsewhere………to follow



Transcript production and processing: before translation intoprotein, the message must be modified

Export from nucleus

………translation

Notes….stability, export and length

transcription start site

5’

3’ 5’

3’ 1 2 3 4 5

Primary RNA transcript

transcription

5’ 3’

Splicing and processing

AAAAACAP

Mature mRNA



5' UTRExon 1 142 bp

Intron 1 130 bp

Exon 2 223 bp

Intron 2 850 bp

Exon 3 262 bp3' UTR

Genomic organisation of the b-globin geneccagggc tgggca taaaa gtcag ggcag agcca tctatt gctt ....

ACATTTG CTTCTG ACACA ACTGT GTTCA CTAGC AACCTC AAACA GACAC C

ATGGTGCATC TGACTC CTGAG GAGAA GTCTG CCGTT ACTGCC CTGTG GGG

CAAGGTG AACGTG GATGA AGTTG GTGGT GAGGC CCTGGG CAGgttggta t

caaggtt acaaga caggt ttaag gagac caata gaaact gggca tgtgg a

gacagag aagact cttgg gtttc tgata ggcac tgactc tctct gccta t

tggtcta ttttcc caccc ttag GCTGCTG GTGGTC TACCC TTGGA CCCAG

AGGTTCT TTGAGT CCTTT GGGGA TCTGT CCACT CCTGAT GCTGT TATGG G

CAACCCT AAGGTG AAGGC TCATG GCAAG AAAGT GCTCGG TGCCT TTAGT G

ATGGCCT GGCTCA CCTGG ACAAC CTCAA GGGCA CCTTTG CCACA CTGAG T

GAGCTGC ACTGTG ACAAG CTGCA CGTGG ATCCT GAGAAC TTCAG Ggtgag

tctatgg gacgct tgatg ttttc tttcc ccttc ttttct atggt taagt t

catgtca taggaa gggga taagt aacag ggtac agttta gaatg ggaaa c

agacgaa tgattg catca gtgtg gaagt ctcag gatcgt tttag tttct t

ttatttg ctgttc ataac aattg ttttc ttttg tttaat tcttg ctttc tttttttt tcttct ccgca atttt tacta ttata cttaat gcctt aacat t

gtgtata acaaaa ggaaa tatct ctgag ataca ttaagt aactt aaaaa a

aaacttt acacag tctgc ctagt acatt actat ttggaa tatat gtgtg c

ttatttg catatt cataa tctcc ctact ttatt ttcttt tattt ttaat t

gatacat aatcat tatac atatt tatgg gttaa agtgta atgtt ttaat a

tgtgtac acatat tgacc aaatc agggt aattt tgcatt tgtaa tttta a

aaaatgc tttctt ctttt aatat acttt tttgt ttatct tattt ctaat a

ctttccc taatct ctttc tttca gggca ataat gataca atgta tcatg c

ctctttg caccat tctaa agaat aacag tgata atttct gggtt aaggc a

atagcaa tatctc tgcat ataaa tattt ctgca tataaa ttgta actga t

gtaagag gtttca tattg ctaat agcag ctaca atccag ctacc attct g

cttttat tttatg gttgg gataa ggctg gatta ttctga gtcca agcta g

gcccttt tgctaa tcatg ttcat acctc ttatc ttcctc ccaca gCTCCT

GGGCAAC GTGCTG GTCTG TGTGC TGGCC CATCA CTTTGG CAAAG AATTC A

CCCCACC AGTGCA GGCTG CCTAT CAGAA AGTGG TGGCTG GTGTG GCTAA T

GCCCTGG CCCACA AGTAT CACTAAGCTCGCT TTCTTG CTGTC CAATT TCT

ATTAAAG GTTCCT TTGTT CCCTA AGTCC AACTA CTAAAC TGGGG GATAT T

ATGAAGG GCCTTG AGCAT CTGGA TTCTG CCTAA TAAAAA ACATT TATTT T

CATTGCA atgatgt atttaa attat ttctg aatat tttac taaa......

What is the structure of a gene?



What is the structure of a gene?



Features of a mature mRNA [messenger RNA = protein coding RNA]

7mG

5’ UTR 3’ UTR PROTEIN CODING SEQUENCE

AAAAAAAAAAAAAAAAAAA

poly A tail

5’ 3’

X

AUG STOP

ALL PROTEIN CODING mRNAs HAVE A POLY A TAIL AT THE 3’ END OF TRANSCRIPT

NOTE: 3’UTR REGIONS ARE NOT UNDER AS MUCH SELECTIVE PRESSURE AS

PROTEIN CODING SEQUENCE



28s

18s

+ve

-ve

Gel electrophoresis of total RNA from a tissue

RNA is

attracted to

+ve

electrode

andseparated

by size;

small RNA

move

quickly

LARGE

SMALL

rRNA

rRNA

mRNA

mRNA is the backgroud ‘smear’

i.e thousands of mRNA molecules of different size [= different size proteins]



Microarrays



What are microarrays?

Tools to measure thousands of simultaneous gene expression levels

Definition:

A gene is said to be expressed if its mRNA or protein are

present in the tissue under study

How do microarrays work?

They rely on the biological principle of complementary hybridisation

AATTATAGCGGAGCGAACGAAG

TTAATATCGCCTCGCTTGCTTC

If we know the mRNA sequence we can build a probe for it with the

complementary DNA sequence





A small probe can detect the presence of its complementary mRNA in a

large population of mRNAs

• the sequence of entire genomes gives us access to thousands [potentially all]

mRNA sequences

• probes can be synthesised for all of the known and predicted mRNAs

• probes are typically 25-70 nucleotides long [25-70mers]

• probes are immobilised on a solid surface [e.g. silicon]

• thousands of probes produced on a silicon base = GeneChip

• if the mRNA from a cell is labelled with a fluorescent dye we can detect which

mRNAS become bound to their complementary probes

• spots of fluorescence on a GeneChip therefore = the genes that are expressed

Mi l tf



Microarray platforms

• spotted cDNA array on nylon membranes

• commercially produced radioactive labelling single channel

• multiple synthesised short oligonucleotides [25mers] on silicon

• commercially produced: Affymetrix

• single channed fluorescent label• between 11 and 20 probe per target gene

• upto 40,000 gene targets/sinle chi

• spotted cDNA or long oligos [70mers] on glass slide

• home grown or commercial

• two channel: simultaneous co-hybridisation of two samples

• two colour fluorescence

Th t t f Aff t i i



The structure of Affymetrix microarrays

•Recap: Affymetric GeneChips [microarrays] contain short

oligonucleotide probes syntesised directly onto a silicontarget using the same technology that Intel uses to make

CPUs

• this allows for a more densely-packed chip but means the

oligos must be shorter

• shorter oligonucleotides may cross-hybridise to the wrong

sequence

• to compensate each given gene [e.g. FGF8] is represented

by 11 different probes scattered across the chip

• the expression of a gene is derived from the overall average

of the reading from the 11 different probes



GeneChip® probe arrays are manufactured through a unique and

robust process, a combination of photolithography and combinational

chemistry

Tens of thousands of probes are printed onto a silicon base

Actual size







Data from an experimentshowing the expression of

thousands of genes on a

single GeneChip® probe

array.



Close up of a GeneChip image

The actual probes have been combined with positive controls that bind

to probes that spell out the chip type and form a border of expression

around the chip

Close up of a GeneChip “feature”



Close up of a GeneChip feature

• 1 feature [18 m square] = the region where probes of a single type are printed

• Each feature contains 100,000s of identical probes

Feature of a gene

that is expressed

Feature of a gene

that is not expressed

Th l l f fl f f t i th



The level of fluorescence of a feature is the sum

of the hybridisation of across the entire region

Affymetrix GeneChips compensate for cross hybridisation by having a



Affymetrix GeneChips compensate for cross hybridisation by having a

Perfect Match and MisMatch Probe feature adjacent to each other

Perfect Match

Mis Match

AATTATAGCGGGGCGAACGAAG

AATTATAGCGGAGCGAACGAAGPM:

MM:

Each individual gene is represented by 11 different probes on an Affymetric microarray



PM

MM

1 2 3 4 5 6 7 8 9 10 11

Probe [25mer] set for 1 gene

Signal = [PM –

MM]-[outliers]/n

i.e. Signal is function of Efficacy of PM/MMOligonucleotide performance NOT absolute expression of gene

Each individual gene is represented by 11 different probes on an Affymetric microarray

The mRNA extracted from a target tissue must be labelled so that it



The mRNA extracted from a target tissue must be labelled so that it

can fluoresce when bound to its complementary probe sequence

1. Extract total RNA from cells or tissue

2. Convert the mRNA only to first strand cDNA

3. An oligo dT primer binds to the poly A tail of mRNA

4. Second strand cDNA is made from first strand

5. The process is done to all the mRNAs from the cell simultaneously

6. Using the double stranded cDNA new complementary DNA are made

7. The new cDNAs contain a biotin label

8. The biotin label can easily be detected using streptavidin and antibodies

9. Biotin-labelled cDNAs [representing the mRNAs] bind to their complementary probes

immobilised on GeneChip



Synthesis of biotin-labelled cRNA rhombomere extracts



Total RNA

Biotin-labelled cRNA

Fragmented cRNA

Bacterial mRNAs spiked

Bacterial cRNAs spiked

Hybridise to arrays

E M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5

SET1 SET2 SET3 SET4

M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5

To minimise the effect of ‘noise’ the probe sets for a



To minimise the effect of noise the probe sets for a

single gene are randomised across the GeneChip

•The intensity and location of the probe features is read by a scanner

•The readings are assimilated into expression values for every single genes







Hybridiseto microarray

Wash offnon-specific

Scan forexpressed genes

Data analysis Confirm differentiallyexpressed genes

Any labelled mRNA that is not tightly-bound to aprobe must be washed off

Ti A Ti B



Tissue A Tissue B

Extract RNA and make labelled probes Extract RNA and make labelled probes

Hybridise to microarray Hybridise to microarray

Genes specific to A and B fluoresce at different positions

Compare the patterns of hybridisation

The output of the scanner is the gene name and a

l ti i l



relative expression value

1

39000

Here: 6 different tissues under study

Microarray data [I.e. the list of genes predicted to be expressed



in a tissue must be confirmed by another technique

• NORTHETN BLOT

• QUANTITATIVE RT-PCR

• IN SITU HYBRIDISATION

• Rnase PROTECTION ASSAY

GeneChips [microarrays] exist for a wide range of species



Yeast

Drosophila

C. elegans Bacteria



Complex statistis are required to analyseand compare a multi GeneChip study

Real differences in gene expression between 2

cells or tissues must be distinguished from

natural or technical variation

To achieve this each cell or tissue in a particular conditionmust be represented more than once

i.e BIOLOGICAL REPLICATESPreferably each condition in MINIMUM OF TRIPLICATE

For example: knockout mouse versus wild typeKO Mouse - use 3 individuals and put on 3 different chips+/+ mouse – use 3 individual and put on 3 different chips

Compare the data between the 2 data sets

Documents

Genes, Genomes & Microarrays