07. Tecnicas de Biologia Molecular

7/28/2019 07. Tecnicas de Biologia Molecular

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Topic1: Nucleic acids

Techniques of Molecular Biology

1.Electrophoresis

2.Restriction3.Hybridization4.DNA Cloning and gene expression

5.PCR6.Genome sequence & analysis


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Organismo Tamao genoma Nmero de genes

H. Influenzae 1.8 Mb 1.700

E.coli 4.2 Mb 4.200

S.cerevisiae 12 Mb 6.000

C. elegans 97 Mb 19.000

A. thaliana 100 Mb 25.000

D. Melanogaster 180 Mb 13.000

Homo sapiens 3.200 Mb 30.000-40.000

Manipulando genes


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1. Gel electrophoresis separates

DNA and RNA moleculesaccording to size, shape andtopological properties

Gel matrix is an inserted, jello-likeporous material that support and

allows macromolecules to movethrough. Agarose and polyacrylamideare two different gel matrices

Electrophoresis


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DNA and RNA molecules are

negatively charged, thus move in thegel matrix toward the positive pole (+)

Linear DNA molecules are separatedaccording to size

The mobility of circular DNAmolecules is affected by theirtopological structures. The mobilityof the same molecular weight DNAmolecule with different shapes is:supercoiled> linear> nicked or relaxed

Electrophoresis


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Fig 20-1: DNA separation by gel electrophoresis

large moderate small After electr


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1. pUC19

2. pUC19/HindIII

3.

4. /HindIII

kb

23.0

9.46.5

4.3

2.3

1 2 3 4

2.0

1% Agarose gel


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To separate DNA of different

size ranges

Narrow size range of DNA: use

polyacrylamide Wide size range of DNA: use

agarose gel

Very large DNA(>30-50kb): usepulsed-field gel electrophoresis

Electrophoresis


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pulsed-field gelelectrophoresis

Switching between twoorientations: the larger theDNA is, the longer it takes

to reorient

Electrophoresis


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Restriction endonucleases cleave

DNA molecules at particular sitesNucleic

acid

Why use endonucleases?

--To make large DNA molecules break intomanageable fragments

--Important tools in recombinant technology

--Have opened the way for gene cloning

--Endonucleases provide protection tobacterial cell against foreign DNA

--Bacterias own DNA is modified by

methylation of bases

Restriction digestion


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Restriction endonucleases: thenucleases that cleave DNA atparticular sites by the recognition of

specific sequences

The target site recognized byendonucleases is usually palindromic.

e.g. EcoRI5.GAATTC..3

.CTTAAG.



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Enzymes are named from bacterial

species that they are derived from

EcoRI is the first enzyme to beisolated from E. colistrain RY13

The enzymes recognise 4-6bp of

DNA and cleave phosphodiester

bond



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To name a restriction

endonuclease:e.g. EcoRI

the 1st

suchenzyme found

Escherichia coli

Species category

R13

strain



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Frequency of the

occurrence of hexamaericsequence:

1/4096 (4-6)

Randomly



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Consider a linear DNA

molecule with 6 copiesofGAATTC:

it will be cut into 7fragments which could

be separated in the gelelectrophoresisby size

(The largest fragment) (The smallestfragment)


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Endonucleases are used to make

restriction map:

e.g. the combination ofEcoRI + HindIII

Allows different regions of one molecule to be

isolate and a given molecule to be identified A given molecule will generate a characteristic

series of patterns when digested with a set ofdifferent enzymes



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Different enzymes recognize theirspecific target sites with differentfrequency

EcoRI Recognize hexameric sequence: 4-6

Sau3A1 Recognize terameric sequence: 4-4

Thus Sau3A1 cuts the same DNA molecule

more frequently



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sticky ends

COHESIVOS

blunt ends

ROMOS



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The 5 protruding ends of are said to besticky because they readily anneal throughbase-pairing to DNA molecules cut with the

same enzyme

Reanneal with itscomplementarystrand or otherstrands with thesame cut



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DNA hybridization can beused to identify specificDNA molecules

Nucleic

acid

Hybridization: the process ofbase-pairing between

complementary ssDNA or RNAfrom two different sources

DNA hybridization


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Probe: a labeled, definedsequence used to searchmixtures of nucleic acids formolecules containing a

complementary sequence

Labeling of DNA or RNA probes


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Labeling of DNA or RNA probes radioactive labeling: display and/or magnify the

signals by radioactivity

Non-radioactive labeling: display and/ormagnify the signals by antigen labeling antibody binding enzyme binding - substrateapplication (signal release)

End labeling: put the labels at the endsUniform labeling: put the labels internally


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Single stranded DNA/RNA

End labeling5-end labeling: polynucleotidekinase (PNK)

3-end labeling: terminaltransferase


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Labeling at both ends by kinase,then remove one end by restriction

digestion

---------------------G---------------------CTTAAp5

5pAATTCG


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Nick translation:

DNase I to introduce random nicks DNA polIto remove dNMPs from 3 to 5 and add new

dNMP including labeled nucleotide at the 3ends.

Hexanucleotide primered labeling:

Denature DNA add random hexanucleotideprimers and DNA pol synthesis of new strandincorporating labeled nucleotide .

Uniformly labeling of DNA/RNA


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Strand-specific DNA probes:

e.g. M13 DNA as template

the missing strand can be re- synthesizedby incorporating radioactive nulceotides

Strand-specific RNA probes:

labeled by transcription

Uniformly labeling of DNA/RNA


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DNA on blot RNA on blot1. Genomic DNA preparation RNA preparation

2. Restriction digestion -

3. Denature with alkali -4. Agarose gel electrophoresis

5. DNA blotting/transfer and fixation RNA

6. Probe labeling 6. Hybridization (temperature)

7. Signal detection (X-ray film or antibody)

Southern and Northern blotting


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Southern blothybridization


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Fingerprinting


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Hibridizacin de coloniaspara identificar clones

que contengan un gen de inters


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bI1 bI2 bI3 bI4 bI5

Northern analysis COB RNAs in S. cerevisiae

mRNA

Pre-mRNAs



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Blot

type

Target Probe Applications

Southern DNA DNA orRNA

mapping genomicclones

estimating genenumbers

Northern RNA DNA orRNA

RNA sizes,abundance,

and expression

Western Protein Antibodies protein size,abundance


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Polymerase chain reactionPCRThe PCR cycle

TemplatePrimersEnzymes

PCR optimization


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PCR

The polymerase chainreaction(PCR) is to used to

amplify a sequence of DNAusing a pair of primers eachcomplementary to one end

of the the DNA targetsequence.

Polymerase chain reaction


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The PCR cycle

Denaturation: The target DNA(template) is separated into two standsby heating to 95

Primer annealing: The temperature isreduced to around 55 to allow theprimers to anneal.

Polymerization (elongation,extension): The temperature is increasedto 72 for optimal polymerization stepwhich uses up dNTPs and required Mg++.



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Template

Any source of DNAthat providesone or more target molecules canin principle be used as a templatefor PCR

Whatever the source of template

DNA, PCR can only be applied ifsome sequence information isknownso thatprimers can bedesigned.



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Primers

PCR primers need to be about 18 to 30 ntlong and have similar G+C contents sothat they anneal to their complementary

sequences at similar temperatures.Theyare designed to anneal on oppositestrands of the target sequence.

Tm=2(a+t)+4(g+c): determine annealingtemperature. If the primer is 18-30 nt,annealing temperature can be Tm5oC



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Degenerate primers:an oligo poolderived from protein sequence.

E.g. His-Phe-Pro-Phe-Met-Lys cangenerate a primer

5-CAY TTYCCN TTYATG AAR

Y= PyrimidineN= any baseR= purine



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Enzymes and PCR Optimization

The most common is Taqpolymerase. It has no 3 to 5proofreading exonuclease activity.Accuracy is low, not good forcloning.

We can change the annealing

temperature and the Mg++concentration or carry out nestedPCR to optimize PCR.



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PCR

amplificacin

DNA

(molcula sencilla)

Muchas

molculas

PCR



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Sntesis por DNA polimerasa

-A T G C A T G C A T G C * *

A CG-T

primers de DNA especficos hibridizan con la cadena quetiene que ser copiada > 18 bp

5 3

PCR



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A CGT-T5 3

PCR



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A CGT-T A5 3

PCR



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A CGT-T A C5 3

PCR



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A CGT-T A CG5 3

PCR



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A CGT-T A CGT5 3

PCR




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A CGT-T A CGTA5 3

PCR



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Despues del primer perodo de sntesis deDNA, tenemos copiada una cadena de DNA

Primer1 Extensin de la cadena

Cadena original de DNA

Cmo produce este proceso una AMPLIFICACIN?

Reaccin en cadena de la polimerasa - PCRPolymerase chain reaction


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Primero, la fusin separa las dos cadenas de DNA a alta

temperatura (~100

C)



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temperatura (~100

C)Luego, usando un segundo primer, se copia la nuevacadena con la DNA polimerasa

Al mismo tiempo, la cadena original se copianuevamente, dado que hay un exceso de Primer 1

2

1

Ahora tenemos dos copias de la molcula original



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Para controlar el proceso, necesitamos controlar la temperatura

Si repetimos el ciclo, tendremos cuatro copias

Reaccin en cadena de la polimerasa - PCR

Fusin95C

Hibridizacin50-60C

Extensin deLa cadena

75C

Primer Ciclo

2do Ciclo95C

50 - 60C75C

Mltiples ciclos darn un incremento exponencial enel nmero de copias



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temperatura (~100

C)



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Template

Primers

Enzymes

Fig.Steps of PCR


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ADN polimerasa termoestable

Oligonucleotidos iniciadores (primers)

Desoxiribonucletidos trifosfatados (dNTPs)

Cationes divalentes

Buffer (para mantener el pH)

Cationes divalentes

ADN molde (Templado)

PCR



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And more PCR


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And more PCR


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ADN polimerasas termoestables

Llevan a cabo la sntesis de ADN dependiente deltemplado.

Estabilidad Taq: 9 min at 97C, Pwo >2 hr a 100C Fidelidad Taq: baja, Pfu: alta

Algunas presentan actividad transferasa terminal en elextremo 3, ej. Taq agrega una A al extremo 3,especialmente si en el extremo hay una C. Pwo y Tligeneran extremos romos

Cantidad usada = 5 x 1012 molculas (1.5 unidades) La m

s com

nmente usada = Taq ADN polimerasa

TaqThermus aquaticus,Pwo Pyrococcus woesei, PfuPyrococcus furiosus, Tli Thermococcus littoralis

PCRPolymerase chain reaction


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Oligonucletidos iniciadores

(primers)

Se conoce su secuencia

Es el factor mas importante para laeficiencia y la especificidad del proceso

Deben estar presentes en exceso (1013 =30 cycles, 1 kb)

Requieren de un cuidadoso diseo Reglas de diseo:

(a) longitud = 18-25

(b) Contenido de G+C entre 40-60%

PCR



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Evitar las secuencias repetidas

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553

3

Dmero de primer

PCR

3repetido

5-NNNNNNNNNNNNNTATA-35-NNNNNNNNNNNNNTATA-3

5-NNNNNNNNTATA-33-ATATNNNNNNNN-5

PCR



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no hay extensin

PCR

5repetido

5-TATANNNNNNNNNNNNN-35-TATANNNNNNNNNNNNN-3

5-TATANNNNNNNN-33-NNNNNNNNATAT-5

55

33



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5

5

3

3

Productos de PCR no deseados

PCR

Formacin de horquillas

5-NNNNNNNNNNNGCATGC-35-NNNNNNNNNNNNNNNNN-3

5-NNNNNNNNNNNGCA3-CGT

5

3



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Oligonucletidos iniciadores

Se conoce su secuencia

Es el factor mas importante para la eficiencia y laespecificidad del proceso

Deben estar presentes en exceso (1013 = 30 cycles, 1kb)

Requieren de un cuidadoso diseo

Reglas de diseo:

(a) longitud = 18-25(b) Contenido de G+C entre 40-60%

(c) Evitar las secuencias repetidas

(d) Valores de Tm de no ms de 5C

uno del otro

PCRolymerase chain reaction


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ADN molde (templado) Puede ser ssDNA o dsDNA (simple o

doble cadena)

ADN circular y cerrado es levemente

menos efectivo que el ADN lineal Usualmente se utilizan varios miles de

copias, ej: 1 g de humano, 10 ng delevadura, 1 ng de bacteriano o 1 pg de

plasmdico Se puede amplificar a partir de una sola

molcula de ADN molde, pero lascondiciones deben estar muy

optimizadas



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El ciclo de PCR Desnaturalizacin - 94-95C por 45 segundos si G+C < 55%

Temperatura de hibridizacin (Annealing) debe ser calculadao determinada empiricamente para cada par de primers

- demasiado alta = poco o nada de producto

- demasiado baja = annealing no especfico= prod.incorrectos

Extensin - a la temperatura ptima de la ADN polimerasautilizada

ej.: 72

C para Taq1 min/kb de longitud, dado que la Taq polimeriza 2000 pb/min

solo a partir del 3r ciclo son producidos ADN duplex de lalongitud deseada, los cuales a partir de all se tornan en elproducto principal

PCRolymerase chain reaction


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Variantes de la PCR

Touchdown PCR

Colony PCR

Multiplex PCRHot start PCR

Nested PCR

Inverse PCR

Long PCR



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Diagnstico

So much DNA that you

can see it with staining!

Medicina Forense: Cebadores para amplificar areas con


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M C p p f

VNTRs (Variable Number Tandem Repeats), que difieren en

diferentes cromosomas y en individuos diferentes


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Three areasamplified to

generate a DNAfingerprint


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Multiplex PCR

Describe una PCR en la cual haypresentes mltiples pares de primers(hasta 8) lo que da una serie de

productos. Los mismos pueden versecomo mltiples bandas en un gel deagarosa

Multiplex PCR es frecuentemente usada

en diagnstico mdico Ahorra templado, tiempo y gastos

Requiere una cuidadosa optimizacin

PCR




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Nested PCR

A veces 1 ronda de PCR no da un productonico a partir de un templado complejo,apareciendo un bandeo inespecfico

Se puede resolver utilizando un segundo parde primers que hibriden un poco masinternamente que los primeros

Realizar una segunda ronda de PCR usando elproducto de la primera (bandeo inespecfico)

Rinde un producto nico porque solo elfragmento correcto de ADN posee los sitioscorrectos de hibridizacin para el segundo parde primers

PCR



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Nested PCR

2da PCR

Bandeo de ADN

1era PCR

ADN especfico

PCR

Clonamiento


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Clonamiento



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Clonado con PCR: clonado T-A

3-AA-3

T-3'

3'-T

A A

T-3'

3'-T

Ligamiento con extremo adhesivo de 1 base = 50 veces mejorque ligamiento con extremos romos

ligamiento

Producto de PCRa partir de Taq

Vector con cola-T


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Mutagnesis por PCR

Introduce cambios de secuencia dentrode fragmentos (clonados) de ADN

El mtodo de extensin solapada

requiere 2 primers mutagnicos y otros2

Amplifica un fragmento 5 y unfragmento 3 que se solapan. Ambos

portan la mutacin Usa los productos en otra reaccin para

producir el ADN mutado de longitudcompleta




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Para amplificar copias de cDNA de RNA

Es especialmente til cuando solose dispone depequeas cantidades de RNA

Frecuentemente usada para amplificar genesespecficos (como cDNAs) si algo de su secuencia esconocida

Requiere primer antisense y un DNA polimerasadependendinte de RNA

Puede ser usada para construir bibliotecas de cDNA

Primero se hace un cDNA a partir del templado deARN

Luego se usa un segundo primer (sense) para hacerel duplex de cDNA por PCR

RT-PCR = PCR con transcriptasa reversa



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RT-PCR

AAAAAAAAAA-3

TTTTTTTTTT-5

Transcripcin reversa

PCR usando

GSP+GSP1

AAAAAAAAAA-3

TTTTTTTTTT-5

mRNA

(sense)

Primer Antisense:

oligo(dT)o

1ra cadena cDNA

GSP1 (sense)

GSP (antisense)

GSP1

GSP

Requiere el conocimiento de la secuencia para disear GSP and GSP1


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SequencingNucleic

acid

Two ways for sequencing:

1. DNA molecules(radioactively labeled at 5termini) are subjected to 4regiments to be brokenpreferentially at Gs, Cs, Ts,

As, separately.2. chain-termination method


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Chain-termination method ddNTPs are chain-terminating nucleotides:

the synthesis of a DNA strand stops when addNTP is added to the 3 end


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The absence of 3-hydroxyl lead to theinefficiency of the nucleophilic attack onthe next incoming substrate molecule


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DNA synthesis abortsat a frequency of 1/100every time thepolymerase meets addGTP

Tell from the gel theposition of each G

DNA sequencing gel


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4 systems with dNTP+ddGTP, dNTP+ ddATP dNTP+ ddCTP, d NTP+

ddTTP separately

read the sequencing

gel to get thesequence of the DNA


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Shortgun sequencing a bacterial genome

The bacterium H. influenzae was thefirst free-living organism to have a

complete genome sequenced andassembled.

This organism is chosen as itsgenome is small (1.8Mb) andcompact.

NUCLE

IC

ACIDS

It h l h d i t


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Its whole genome was sheared into manyrandom fragments with an average length of

1kb.

This pieces are cloned into a plasmid vector.And these clones are sequenced respectively.

All these sequence information are loadedinto the computer. The powerful program will

assemble the random DNA fragment basedon containing matching sequence, forming asingle continuous assemble, called a contig.

To ensure every nucleotide in the genome


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To ensure every nucleotide in the genomewas captured in the final genome assemble,30000~40000 clones are needed, which isten times larger as the genome. This iscalled 10sequence coverage.

This method might seem tedious, but itsmuch faster and cheaper than thedigestion-mapping-sequencing method. As

the computer is much faster at assemblingsequence than the time required to mapthe chromosome.

permits a partial


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The shortgun strategy permits a partialassembly of large genome sequence

If we want to sequence a much largerand more complicate eukaryotic

genome using the shortgun strategy.What can we do?

Firstly, libraries in different level shouldbe constructed.

NUCLE

ICACIDS


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91 DNA microarrays


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92 Microarreglos de DNA


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(DNA microarrays) Microarreglos de DNA: basado en

las propiedades de hibridizacin de

los Acidos Nucleicos para monitoreara escala genmica la abundancia deDNA or RNA en distintos tipos de

clulas.

Nucleic acid


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Nucleic acidhybridization

Microarrays


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Microarrays Place probes from many different

genes on a glass microscope slide,

then hybridize to cDNA made frommessenger RNA isolated from atissue. You see which genes areactive in that tissue.

Mostly done with 60mers: 60 baseslong, synthetic oligonucleotides,made using sequence informationfrom the genes.

cDNA is fluorescently labeled Often 2 conditions are compared

(control and experimental), usingred and green fluorescent tags.

Semi-quantitative

Can also be used to screen for DNA

mutations.


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The processBuilding the chip:

MASSIVE PCR PCR PURIFICATION

AND PREPARATION

PREPARING

SLIDES

PRINTING

RNA preparation:

CELL CULTURE

AND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the

chip:

ARRAY HYBRIDIZATION

PROBE LABELING DATA ANALYSIS

POSTPROCESSING


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The arrayer


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Ngai Lab arrayer , UC Berkeley

The arrayer

Print-tip head

Pins collect cDNAfrom wells


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384 wellplate

Contains cDNA

probesGlass Slide

Array of bound cDNA probes

4x4 blocks = 16 print-tip groups

Print-tipgroup 6

cDNA clones

Spotted in duplicate

Print-tipgroup 1


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Samplepreparation


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Hybridization

Hybridize for

5-12 hours

Binding of cDNA target samples to cDNA probes on the slide


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LABEL

3XSSC

HYB CHAMBER

ARRAY

SLIDE

LIFTERSLIP

SLIDE LABEL

Humidity Temperature

Formamide

(Lowers the Tm)

Hybridization chamber

Scanning


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Detector

PMT

Image

Duplicatespots

RGB overlay of Cy3 and Cy5


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images

Mic oa a life c le


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Biological

Question

SamplePreparation

DataAnalysis &Modelling

Microarray

Reaction

MicroarrayDetection

Taken from Schena & Davis

Microarray life cyle

Biological questionDifferentially expressed genesS l l di i


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Sample class prediction etc.

Testing

Biological verification

and interpretation

Microarray experiment

Estimation

Experimental design

Image analysis

Normalization

Clustering DiscriminationR, G

16-bit TIFF files

(Rfg, Rbg), (Gfg, Gbg)


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Part II proteins

Specific proteins can be purified from


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cell extracts

The purification of individual proteins iscritical to understanding their function.(why?)

Although there are thousands ofproteins in a single cell, each proteinhas unique properties that make itspurification somewhat different fromothers.

proteins


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The purification of a protein isdesigned to exploit its uniquecharacteristics, such as size,charge, shape, and in manyinstance, function.

Separation of proteins on polyacrylamide gels


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Separation of proteins on polyacrylamide gels

The native proteins have neither auniform charge nor a uniformsecondary structure.

If we treat the protein with a strongdetergent SDS, the higher structure isusually eliminated. And SDS confersthe polypeptide chain a uniformnegative charge.

pr

oteins

A d ti t th l i


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And sometimes mercaptoethanol isneed to break the disulphide bond.

Thus, the protein molecules can beresolved by electrophoresis in thepresence of SDS according to thelength of individual polypeptide.

After electrophoresis, the proteins canbe visualized with a stain, such as

Coomassie brilliant blue.

Antibodies visualize electrophoretically-


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p y

separated proteins

The electrophoretically separatedproteins are transferred to a filter. And

this filter is then incubate in a solutionof an antibody to our interested protein.Finally, a chromogenic enzyme is used

to visualized the filter-bound antibody.

pr

oteins

Western Blotting


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Protein denature(SDS)

SDS-PAGE gel

Blot

Blocking (BSA) 1 Ab serum (probe)

Wash

2 Ab serum

Wash Development

Analysis of protein samples by SDSpolyacrylamide gel electrophoresis and Western


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polyacrylamide-gel electrophoresis and Western

blotting

Protein bandsdetected byspecific antibody

SDS-PAGE Western blot

ELISA
http://d/My%20Documents/3EIMMBLOT.MOVhttp://d/My%20Documents/3EIMMBLOT.MOVhttp://d/My%20Documents/3EIMMBLOT.MOVhttp://d/My%20Documents/3EIMMBLOT.MOV


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ELISA (Enzyme-Linked Immunosorbent Assay)

Coating of antigen in microplate Block unbound sites in wells

Apply antibody to wells

Add anti-mouse IgG (conjugate to an enzyme

Substrate reaction for color indication

Purification of a protein requires a


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specific assay To purify a protein requires that you

have an assay that is unique to thatprotein.

In many instance, its convenient touse a measure for the function of theprotein, or you may use the antibody

of the protein. It is useful to monitor the purification

process.

proteins

Proteins can be separated from oneh i l h h


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another using column chromatography

In this approach, protein fractionsare passed though glass columnsfilled with appropriated modifiedsmall acrylamide or agarose beads.

There are various ways columns canbe used to separate proteinsaccording to their characteristics.

proteins

Ion exchange chromatography


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Ion exchange ch omatog aphy

The proteins are separated accordingto their surface charge.

The beads are modified with eithernegative-charged or positive-chargedchemical groups.

Proteins bind more strongly requiresmore salt to be eluted.


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Gel filtration chromatography


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Gel filtration chromatography

This technique separate the proteins onthe bases of size and shape.

The beads for it have a variety ofdifferent sized pores throughout.

Small proteins can enter all of the pores,and take longer to elute; but largeproteins pass quickly.


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Affinity chromatography can facilitate

id i ifi i


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more rapid protein purification

If we firstly know our target protein canspecifically interact with something else,

we can bind this something else tothe column and only our target proteinbind to the column.

This method is called affinitychromatography.

pr

oteins

Immunoaffinity chromatography


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Immunoaffinity chromatography

An antibody that is specific for thetarget is attached to the bead, andideally only the target protein canbind to the column.

However, sometimes the binding is

too tight to elute our target protein,unless it is denatured. But thedenatured protein is useless.

Sometimes tags (epitopes) can be addedto the N- or C- terminal of the protein


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to the N- or C- terminal of the protein,using molecular cloning method.

This procedure allows the modifiedproteins to be purified using

immunoaffinity purification and aheterologous antibody to the tag.

Importantly, the binding affinity canchange according to the condition. e.g. theconcentration of the Ca2+ in the solution.

Immunoprecipitation


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p p

We attach the antibody to the bead,and use it to precipitate a specificprotein from a crude cell extract.

Its a useful method to detect whatproteins or other molecules areassociated with the target protein.

Th i i l h d


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Three principle methods

1. 2-D gel electrophoresis for proteinseparation.

2. MS for the precise determination of aprotein.

3. Bioinformatics technology.

rotein molecules can be directly sequence


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Two sequence method: Edmandegradation &Tandem massspectrometry(MS/MS).

Due to the vast resource ofcomplete or nearly completegenome, the determination of evena small stretch of protein sequenceis sufficient to identify the gene.

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oteins

Ed d d ti


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Edman degradation

Its a chemical reaction in which theamino acids residues aresequentially release for the N-terminus of a polypeptide chain.

Step 1: modify the N-terminal amino withPITC, which can only react with the free -


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, yamino group.

Step 2: cleave off the N-terminal by acidtreatment, but the rest of the polypeptideremains intact.

Step 3: identify the released amino acids byHigh Performance Liquid

Chromatography (HPLC).

The whole process can be carried out in an

automatic protein sequencer


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Tandem mass spectrometry


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Tandem mass spectrometry

MS is a method in which the massof very small samples of a

material can be determined.

Step 1: digest your target protein intoshort peptide.


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Step 2: subject the mixture of the

peptide to MS, and each individualpeptide will be separate.

Step 3: capture the individual peptide

and fragmented into all the componentpeptide.

Step 4: determine the mass of each

component peptide. Step 5:Deconvolution of these data and

the sequence will be revealed.


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Proteomics


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Proteomics

Proteomics is concerned with theidentification of the full set ofproteins produced by a cell or atissue by a particular set ofconditions.

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07. Tecnicas de Biologia Molecular

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