Special Topics in Computer Science: Bioinformatics & Computational BiologBioinformatics & Computational Biology

A special lecture on molecular biology for computer scientists

P G

computer scientists

P I N G G O N G

S E N I O R S C I E N T I S T , P H . D .

B A D G E R T E C H N I C A L S E R V I C E S , L L CB A D G E R T E C H N I C A L S E R V I C E S , L L C

E N V I R O N M E N T A L L A B O R A T O R Y

U S A R M Y E N G I N E E R R E S E A R C H A N DD E V E L O P M E N T C E N T E RD E V E L O P M E N T C E N T E R

Outline

Introduction: what is the goal? Human genomic projects Molecular biology for computational scientists Life from a taxonomy viewpoint Life from a taxonomy viewpoint Cell biology 101 Genetics, genomics and epigenomics

G i l ti Gene expression regulation Biochemistry

Molecular biology technologiesgy g Traditional molecular biology techniques Genomics Proteomics Proteomics Metabolomics

Traditional molecular biology technologies

P l h i ti (PCR) Polymerase chain reaction (PCR) Quantitative PCR

M l l l i Molecular cloning DNA sequencing

Polymerase Chain Reaction (PCR)

PCR is a molecular biology technique to amplify a particular PCR is a molecular biology technique to amplify a particular segment of DNA (target DNA, up to 40 kb in length) across several orders of magnitude, generating thousands to millions of copies of the DNA fragment.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.Th i l d DNA l i f i DNA b d These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.g

Polymerase Chain Reaction (PCR)

The method relies on thermal cycling, i ti f l f t d h ti d consisting of cycles of repeated heating and

cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Primers (short DNA fragments) containing Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key

A strip of eight PCR tubes, each containing a 100 μl reaction mixture) y

components to enable selective and repeated amplification.

As PCR progresses, the DNA generated is i lf d l f li i itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplifiedamplified.

Thermal cycler

Polymerase Chain Reaction (PCR)

A basic PCR set up requires the following PCR requirementsA basic PCR set up requires the following components and reagents: DNA template that contains the DNA region

(target) to be amplified Two primers that are complementary to the 3'

PCR requirements Magnesium chloride: .5-

2.5mM Buffer: pH 8.3-8.8 dNTPs: 20-200µM Two primers that are complementary to the 3

ends of each of the sense and anti-sense strand of the DNA target.

Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.

dNTPs: 20-200µM Primers: 0.1-0.5µM DNA Polymerase: 1-2.5

units Target DNA: 1 µg

Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.B ff l ti idi it bl h i l

Target DNA: 1 µg PCR thermal cycles

Denaturalization: 94°-95°CP i A li ° Buffer solution, providing a suitable chemical

environment for optimum activity and stability of the DNA polymerase.

Divalent cations, magnesium (Mg2+) or manganese ions

Primer Annealing: 55°-65°C

Extension of DNA: 68°-72°

Number of Cycles: 25 40manganese ions Monovalent cation potassium (K+) ions

Number of Cycles: 25-40

Polymerase Chain Reaction (PCR)

A recipe for PCR master mix (1 reaction): Water 4.42 10x PCR Buffer (minus Mg2+) 1 10 mM dNTP mix 0.2 50 mM MgCl2 0.3

F d i ( M) Forward primer (2.5 µM) 1 Reverse primer (2.5 µM) 1 Platinum Taq DNA polymerase (5 U/µl) 0.08 Template DNA (~20 ng/µl) 2 TOTAL 10 µl TOTAL 10 µl

A protocol for PCR thermal cycling: 94 degree for 5 min (hot start)

l f 35 cycles of 94 degree for 30 seconds (denaturation) 56 degree for 45 seconds (annealing) 68 degree for 45 seconds (elongation)

68 degree for 10 min8 g 4 degree forever

Visualization by Gel Electrophoresis

Ethidium bromide stained Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate p 3successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA f f d fi d DNA fragments of defined length for sizing the bands in the experimental PCRs.

• DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive pole (anode)pole (anode).

• An agarose gel is used to slow the movement of DNA and separate by size.

H

O2

+-DNA

+-

Power • Polymerized agarose is porous,

Scanning Electron Micrograph of Agarose Gel (1×1 µm)

allowing for the movement of DNA

Staining the Gel• Ethidium bromide binds to DNA and fluoresces under UV light, ll i th i li ti f DNA G lallowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel and/or running bufferbefore the gel is run or the gel can be stained after it has run.

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

Safer alternatives to Ethidium Bromide

Methylene Blue

BioRAD - Bio-Safe DNA Stain

Ward’s - QUIKView DNA Stain

Carolina BLU Stain

Invitrogen’s SYBR Green I Invitrogen s SYBR Green I…othersad antages di d tadvantagesInexpensiveLess toxicNo UV light requiredN h d t di l

disadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time

No hazardous waste disposal

Visualization using SYBR Green I

SYBR Green I preferentially binds to double-stranded DNA, but will stain single stranded DNA and RNA with lower performancestain single-stranded DNA and RNA with lower performance.

DNA ladderwells

2 000 bpPCR

250

1,500 1,000

500 750

2,000 bpPCRProduct

250+ - - - - + + - - + - +

Traditional molecular biology technologies

P l h i ti (PCR) Polymerase chain reaction (PCR) Quantitative PCR

M l l l i Molecular cloning DNA sequencing

Quantitative PCR (QPCR)

QPCR (real-time quantitative PCR, RT-QPCR)QPCR (real time quantitative PCR, RT QPCR) Definition: Fluorescence-based real-time detection/monitoring of

amplification products through the use of a DNA-binding dye or hybridization probe.

Purpose: To estimate the initial quantity (copy number) of specific template DNA by measuring the number of cycles required to reach a set level of product.

The target can be nucleic acids (RNA or DNA) The target can be nucleic acids (RNA or DNA). Reverse transcription quantitative polymerase chain reaction (RT-

QPCR) is a laboratory technique commonly used in molecular biology where a RNA strand is first reverse transcribed into its DNA

l t ( l t DNA DNA) i th complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using PCR. Reverse transcription PCR is not to be confused with real-time quantitative PCR, which is also sometimes abbreviated as RT-QPCR.

QPCR vs. PCR

Real-Time PCR Traditional PCR h f l d C d Overview Measures PCR amplification as it occurs. Measures the amount of accumulated PCR product at

the end of the PCR cycles.

Quantitative?

Yes, because data is collected during the exponential growth (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of

No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you 'semi quantitative' resultsp y p p

template nucleic acid. can give you semi-quantitative results.

•Quantitation of Gene Expression•Microarray Verification•Quality Control and Assay Validation•Pathogen detection

Amplification of DNA for:

•Sequencing•Genotyping

ApplicationsPathogen detection

•SNP Genotyping•Copy Number Variation•MicroRNA Analysis•Viral Quantitation•siRNA/RNAi experiments

Genotyping•Cloning

Summary

Advantages of Real-Time PCR•Increased dynamic range of detection•No post-PCR processing•Detection is capable down to a 2-fold change•Collects data in the exponential growth phase of PCR

Disadvantages of Traditional PCR•Poor Precision•Low sensitivity•Short dynamic range < 2 logs•Low resolution

•An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated•The cleaved probe provides a permanent record amplification of an amplicon

•Non-Automated•Size-based discrimination only•Non-quantitative•Post-PCR processing

QPCR Publications

Publications on qPCR have grown a lot and its graph looks like a qPCR amplification

Commercial real-time PCR machine

ABI 7900HT Fast Real-Time PCR System

QPCR Chemistry

TaqMan chemistry

Quantitative PCR (QPCR)

Data collection

Quantitative PCR (QPCR)

Data analysis:Th h ld C l (C ) Th i hi h h Threshold Cycle (CT): The point at which the fluorescence rises appreciably above background Threshold can be placed anywhere in the exponential (log-linear)

phasephase. After baseline subtraction, a threshold line is set empirically or by

a statistical calculation at a fluorescence value above background.

QPCR: amplification efficiency

th h ldthreshold

Serial 10-fold dilutions

QPCR: absolute quantification

http://en.wikipedia.org/wiki/DNA_cloning

‘copy number’ reference gene experimental

Dilution curve reference gene

py g p

‘copy number’ reference gene control

QPCR: relative quantification

QPCR: relative quantification

IL1-b vit

RPLP0 con

IL1-b conRPLP0 vit

RPLP0 con

Ct/c = 9 70 Ct/c = target - refcontrol

Ct/c = 9.70

IL1-b con

av =19.93 av =29.63

IL1 b i Ct/e = target - refexperiment IL1-b vit

RPLP0 vit

Ct/e = -1.7 Ct/e target refexperiment

av =18.03

Difference = Ct/c-Ct/e= Ct

av =19.80 = 9.70-(-1.7)= 11.40

Traditional molecular biology technologies

P l h i ti (PCR) Polymerase chain reaction (PCR) Quantitative PCR

M l l l i Molecular cloning DNA sequencing

Molecular Cloning: definition

TechniqueTechnique involvinginvolving thethe insertioninsertion ofof aafragmentfragment ofof foreignforeign DNADNA intointo aa vectorvectorfragmentfragment ofof foreignforeign DNADNA intointo aa vectorvectorcapablecapable ofof replicatingreplicating autonomouslyautonomously inin aah th t llll ( ll( ll E h i hiE h i hi lili)) G iG ihosthost cellcell (usually(usually EscherichiaEscherichia colicoli)).. GrowingGrowingthethe hosthost cellcell allowsallows thethe productionproduction ofofmultiplemultiple copiescopies ofof thethe insertedinserted DNADNA forfor useuseinin aa varietyvariety ofof purposespurposes..

Molecular Cloning: requirements

ForeignForeign DNADNA ForeignForeign DNADNA

HostHost organismorganism

VectorVector DNADNA forfor cloningcloning

MeansMeans ofof insertinginserting foreignforeign DNADNA intointo thethe vectorvectorgg gg

MethodMethod ofof placingplacing thethe inin vitrovitro modifiedmodified DNADNAintointo thethe hosthost cellcellintointo thethe hosthost cellcell

MethodsMethods forfor selectingselecting and/orand/or screeningscreening cellscells thatthatcarrycarry thethe insertedinserted foreignforeign DNADNA

Molecular Cloning: requirements

ForeignForeign DNADNA ForeignForeign DNADNA

HostHost organismorganism

VectorVector DNADNA forfor cloningcloning

MeansMeans ofof insertinginserting foreignforeign DNADNA intointo thethe vectorvectorgg gg

MethodMethod ofof placingplacing thethe inin vitrovitro modifiedmodified DNADNAintointo thethe hosthost cellcellintointo thethe hosthost cellcell

MethodsMethods forfor selectingselecting and/orand/or screeningscreening cellscells thatthatcarrycarry thethe insertedinserted foreignforeign DNADNA

Molecular Cloning: requirements

PolymerasesPolymerasesRestriction Restriction EndonucleasesEndonucleasesPolymerasesPolymerases

DNA Polymerase DNA Polymerase –– catalyzes the polymerization of catalyzes the polymerization of deoxyribonucleotidesdeoxyribonucleotides along the template strandalong the template stranddeoxyribonucleotidesdeoxyribonucleotides along the template strandalong the template strand

DNADNA--dependent RNA Polymerasedependent RNA Polymerase

NucleasesNucleases

Enzymes capable of cleaving the phosphodiester bonds Enzymes capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acidsbetween nucleotide subunits of nucleic acids

Other Modifying EnzymesOther Modifying EnzymesOther Modifying EnzymesOther Modifying Enzymes

LigasesLigases

f f h h di th h di t b d t j i t i f DNAb d t j i t i f DNAforms forms phosphodiesterphosphodiester bonds to join two pieces of DNAbonds to join two pieces of DNA

utilizes ATP in the presence of Mgutilizes ATP in the presence of Mg++++

KinasesKinases

transfers phosphate groups from donor moleculestransfers phosphate groups from donor moleculestransfers phosphate groups from donor moleculestransfers phosphate groups from donor molecules

phosphorylasephosphorylase

PhosphatasesPhosphatasespp

catalyzes the removal of 5’catalyzes the removal of 5’--phosphate residuesphosphate residues

ForeignForeign DNADNAgg

PCRPCR productproduct

genomicgenomic DNADNAgenomicgenomic DNADNA

complementarycomplementary DNADNA (cDNA)(cDNA)

HostHost organismorganism

b t i lb t i l h th t EE lilibacterialbacterial hosthost –– EE.. colicoli

eukaryoticeukaryotic hosthost –– yeastyeast ((SaccharomycesSaccharomyces cerevisiae)cerevisiae)

otherother hostshosts –– otherother yeasts,yeasts, insectinsect cells,cells, etcetc..

VectorVector DNADNA

DNADNA moleculemolecule thatthat functionsfunctions asas aa “molecular“molecular carrier”carrier”thatthat carrycarry thethe DNADNA ofof interestinterest intointo thethe hosthost cellcell && facilitatesfacilitatesitit li tili tiitsits replicationreplication..

PlasmidsPlasmids –– usedused inin cloningcloning smallsmall segmentssegments ofof DNADNA ((1010--1515 kb)kb)

BacteriophageBacteriophage λλ –– usedused inin cloningcloning largerlarger segmentssegments ofof DNADNA(~(~2020 kb)kb)

CosmidsCosmids –– plasmidsplasmids containingcontaining DNADNA sequencessequences ((coscos)) fromfrombacteriophagebacteriophage λλ usedused toto cloneclone largerlarger fragmentsfragmentsofof upup toto 4545 KbKb

•• small circular dsDNA that autonomously replicates small circular dsDNA that autonomously replicates apart apart from the chromosome of the host cellfrom the chromosome of the host cellpp

•• “molecular parasites”“molecular parasites”

•• carry one or more genes some of which confer carry one or more genes some of which confer resistance to certain antibioticsresistance to certain antibiotics

•• origin of replication (ORI) origin of replication (ORI) ------ a region of DNA that a region of DNA that allows multiplication of the plasmid within the allows multiplication of the plasmid within the hosthost

smallsmall sizesize

DesirableDesirable propertiesproperties ofof plasmidsplasmids::

smallsmall sizesize

knownknown DNADNA sequencesequence

highhigh copycopy numbernumber

aa selectableselectable markermarker aa selectableselectable markermarker

aa secondsecond selectableselectable genegene

largelarge numbernumber ofof uniqueunique restrictionrestriction sitessites

MeansMeans ofof insertinginserting foreignforeign DNADNA intointo thethe vectorvector

LigationLigation ofof thethe DNADNA intointo thethe linearizedlinearized vectorvector

RequirementsRequirements forfor aa ligationligation reactionreaction::•• two or more fragments of DNA (blunt/cohesive)two or more fragments of DNA (blunt/cohesive)

•• buffer containing ATPbuffer containing ATP

RequirementsRequirements forfor aa ligationligation reactionreaction::

buffer containing ATPbuffer containing ATP

•• TT4 4 DNA DNA ligaseligase

MethodMethod ofof placingplacing thethe inin vitrovitro modifiedmodified DNADNAp gp gintointo thethe hosthost cellcell

TransformationTransformation intointo thethe hosthost cellcellTransformationTransformation intointo thethe hosthost cellcell

•• bacterial cells take up naked DNA moleculesbacterial cells take up naked DNA molecules

•• cells are made “competent”cells are made “competent”

•• cells treated with icecells treated with ice--cold CaClcold CaCl22 then heatthen heat--shockedshocked

•• efficiency of efficiency of 101077 to to 101088 transformed colonies/transformed colonies/μμg DNAg DNA

•• maximum transformation frequency of maximum transformation frequency of 1010--33maximum transformation frequency of maximum transformation frequency of 1010

ElectroporationElectroporation ofof thethe DNADNA intointo thethe hosthost cellcell

•• “electric field“electric field--mediated membrane mediated membrane

ion.

jpg

permeabilization” permeabilization”

•• high strength electric field in the high strength electric field in the

p/im

g/el

ectro

pora

ti

presence of DNApresence of DNA

•• protocols differ for various speciesprotocols differ for various species

.ac.

jp/re

sear

ch/e

p

•• efficiencies of 10efficiencies of 1099 per per μμg DNA (3 kb) g DNA (3 kb) & 10& 1066 (136 kb)(136 kb)

p://b

me.

pe.u

-toky

oht

tp

MethodsMethods forfor selectingselecting and/orand/or screeningscreening cellscells thatthatcarrycarry thethe insertedinserted foreignforeign DNADNA

SelectionSelection refersrefers toto applicationapplication ofof conditionsconditions thatthatSelectionSelection refersrefers toto applicationapplication ofof conditionsconditions thatthatfavorsfavors thethe growthgrowth ofof cellscells oror phagesphages thatthatcarrycarry thethe vectorvector oror vectorvector andand insertinsertcarrycarry thethe vectorvector oror vectorvector andand insertinsert..

•• antibiotic resistanceantibiotic resistance

•• nutrient requirementsnutrient requirements

ScreeningScreening allowsallows allall cellscells toto grow,grow, butbut teststests thetheScreeningScreening allowsallows allall cellscells toto grow,grow, butbut teststests thetheresultingresulting clonesclones forfor thethe presencepresence ofof thethe insertinsertinin thethe vectorvector..

•• antibiotic resistance/sensitivityantibiotic resistance/sensitivity

•• nutrient requirementsnutrient requirements

•• blueblue--white selection (white selection (ββ--galactosidase)galactosidase)

•• specific (hybridization, antibodies, PCR)specific (hybridization, antibodies, PCR)

PositivePositive selectionselection

disruptiondisruption ofof thethe lacZlacZαα--ccdBccdB genegene

permitspermits growthgrowth ofof positivepositive recombinantsrecombinants

DNADNA isolationisolation forfor::

makingmaking probesprobes

restrictionrestriction mappingmapping

sequencingsequencingq gq g

reintroductionreintroduction intointo organismorganism

EstablishmentEstablishment ofof collectionscollections:: DNADNA LibrariesLibraries

FurtherFurther molecularmolecular studiesstudies:: productionproduction ofof specialspecialproteinsproteins

Traditional molecular biology technologies

P l h i ti (PCR) Polymerase chain reaction (PCR) Quantitative PCR

M l l l i Molecular cloning DNA sequencing

DNA Sequencing

Maxam-Gilbert Sequencing(chemical sequencing)

DMS FA H H+S

GG

GA

CT

CC

GG

GG

AG

A

TC

TC

C

CC

Maxam-Gilbert sequencing is performed by A

A CT

chain breakage at specific nucleotides.

Maxam-Gilbert Sequencing(chemical sequencing)

G G+A T+C C

3′AAGG G+A T+C C CAAC

Longer fragmentsA

GTGCShortest fragments

GAG5′

G

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

Chain-termination method(Sanger sequencing)

Questions?