what is pcr? pcr

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Sep 17, 2010

PCRPCRthe the swissswiss armyarmy knifeknife

Claudia Stäubert, Institute for biochemistry

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.

What is PCR?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

What is PCR?Why Polymerase?

It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

What is PCR?Why chain?

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

What is PCR?The „reaction“ components

THERMOCYCLERPCR tube

What is PCR?The „reaction“

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How PCR works PCR – swiss army knifeAllele-specific PCR (SNP detection)Assembly PCR (artificial synthesis of long DNA sequences)Asymmetric PCR (preferential amplification of one DNA strand)Helicase-dependent amplification (usage of constanttemperture – no cycling)Hot-start PCR (reduction of non-specific amplification)Intersequence-specific PCR (ISSR) (DNA fingerprinting)Inverse PCR (used to identify flanking sequencesaround genomic inserts)Ligation-mediated PCR (small DNA-linkers ligated to DNA, multiple primer annealing to linkers) – e.g. genomewalking

PCR – swiss army knifeMethylation-specific PCR (MSP) (for detection of CpGislands in genomic DNA)Miniprimer PCR (thermostable polymerase that canextent „smalligos“ 9-10bp)Multiplex Ligation-dependent Probe Amplification(MLPA) (amplification of multiple targets with a singleprimer pair)Multiplex PCR (multiple primer sets within a single PCR reaction – prodution of amplicons of varying size)Nested PCR (increased specifity of amplification, 2 primer sets in 2 successive PCRs)Overlap-extension PCR (genetic engineering technique– insertion of alterations)

PCR – swiss army knife

Quantitative PCR (measurement of quantity of a PCR productin real-time using fluorescent dyes or fluorophore containingDNA probes)

PCR – swiss army knifeRandom-mutagenesis PCR (introduces randonlymutations)Reverse Transcription PCR (amplification of DNA fromRNA using reverse transcriptase)Site-directed mutagenesis PCR (introduces mutationsat specific sites)Solid phase PCR (either primers or probes arecovalently linked)Thermal asymmetric interlaced PCR (TAIL PCR) (isolation of unknown sequence flanking a knownsequence)Touchdown PCR (gradual reduction of annealingtemerature as PCR cycling progresses)

PCR ApplicationsMolecular identification

Classification of organismsMolecular archeologyMolecular epidemiologyMolecular ecologyDNA fingerprinting (forensics)SNP Genotyping (population analysis)Mutation detection and characterizationGenetic diagnosis (e.g. Pre-natal diagnosis)Organism detection (pathogenes, e.g. virus, bacteria)

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PCR ApplicationsSequencing

Mammalian/human genomesancestral genomes (Neanderthal)biomolecular computingTaxonomic relationships/phylogenies

PCR ApplicationsGenetic engineering

Molecular cloningSite-directed/random mutagenisisGene expression studies (RNA)



Classification of organism

Relation to otherspeciesSimilaritiesDifferences

BUT what to do whenyou have onlyFossilsTrace amountssmall organisms

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SEQUENCING


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PCR-based locus specific RFLP

specific locus amplified by PCRPCR product cut by restrictionendonucleasesvariation in fragment length detectable bygel electrophoresis

Classification of organsims

REP-PCR

repetitive extragenic palindromic (REP) elements: 38bp sequence, 6 degeneratepositions and 5bp variable loopenterobacterial repetitive intergenicconsensus (ERIC): 126bp elemts

described for enteric bacteriaamplification patterns used to discriminatebetween strains

RAPD assay

random amplified polymorphic DNA (RAPD)short (9-10bp) random sequence primer

number and location of random primer sites vary for different strains of bacteriaspecific pattern on gel

CFLPcleavase fragment length polymorphism(CFLP)Cleavase I: thermostable, engineeredendonuclease, recognizes structuresformed by base-paired DNA at elevatedtemperatures95°C 60°C single strand DNA – self-base pairing phase unique hairpinstructures cut by Cleavase Iunique pattern on gel

AFLP

amplified fragment lenghth polymorphism(AFLP)genome fingerprinting techniquedigestion of total cellular DNA with one or morerestriction enzymes and ligation of restrictionhalf-site specific adaptors to all restrictionfragments.

selective amplification of a subset of theseDNA fragmentselectrophoreses analyses of specific band pattern

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AFLP PCR ApplicationsMolecular identification


Molecular archaeology

use DNA or mtDNA (mitochondrial DNA) to determine species of natural archaeological finds as well as determine blood lines and/or sex of animal or human remainsreconstruction of subsistence and related cultural activities possible



Molecular epidemiology

“A science that focuses on the contribution of potential genetic and environmental risk factors, identified at the molecular level, to the etiology, distribution and prevention of disease within families and across populations"

Descriptive and analytical studies to evaluate host/environmental interactions in disease Development of prevention strategies for the control of bacterial, parasitic and viral disorders through molecular diagnosis Prevention of non-communicable diseases and genetic disorders by assessing risk and identifying susceptible individuals through genetic screening

Detection of pathogenes

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Molecular ecology

Field of evolutionary biology that is concerned with applying molecular population genetics, molecular phylogenetics, and more recently genomics to traditional ecological questions (e.g., species diagnosis, conservation and assessment of biodiversity, species-area relationships, and many questions in behavioralecology).


Classification of organismsMolecular archeologyMolecular epidemiologyMolecular ecologyDNA fingerprinting (forensics, paternity testing)SNP Genotyping (population analysis)Mutation detection and characterizationGenetic diagnosis (e.g. Pre-natal diagnosis)Organism detection (pathogenes, e.g. virus, bacteria)

DNA fingerprinting (DNA typing/profiling)Even though two unrelated humans differ in their DNA only by 0.1 to 0.2% there are still up to 6 million basepair differences

It is these differences that are used to create a unique DNA “fingerprint” also known as DNA profile

Human genetics variationPrimarily two types of genetic mutation events create all forms of variations:

Single base mutation which substitutes one nucleotide for another

Single Nucleotide Polymorphisms (SNP)Insertion or deletion of one or more nucleotide(s)

Tandem Repeat PolymorphismsInsertion/Deletion Polymorphisms

4 criteria for selecting useful DNA fingerprinting markers1. Markers should be polymorphic (so that they are

informative)2. Markers should be single locus (so that they occur in

only one location in the genome and there is no ambiguity about their number or position)

3. Markers should be located on different chromosomes (so that the markers are independent)

4. For some applications such as the study of population size and changes over time, markers should beneutral (so that they are not correlated with selection or adaptation; unless selection of adaptation are to be studied; selection confounds estimates of population size parameters)

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Good DNA markers

Repeated DNAMinisatellites (VNTRs = variable number tandem repeats)

Repeated units (5-50 times) of 10 to several 100 bpGC-richinherited codominantly

Microsatellites (STRs = short tandem repeats)Repeated units of 2-6 bp

Minisatellite repeat (VNTR)

http://www.usask.ca/biology/rank/316/genomics/genomics.htm

One Mechanism of VNTR Creation PCR amplification of VNTR

This process requires primers that anneal just outside the VNTR

http://www.genelex.com/paternitytesting/paternityslide2.html

Single-locus minisatelliteVNTRs generates at most two bands

Though not as unique as multi-locus VNTRs they are simple to use

Multiple single-locus VNTRs are used to give a DNA fingerprint

Single-Locus VNTR Multi-Locus VNTR

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Microsatellites

short tandem repeats (STR) or simple sequence repeat (SSR)Heterozygote

Male 5’-TAATAATAATAATAATAATAA----3’Female 5’-TAATAATAATAATAATAATAATAA-3’

HomozygoteMale 5’-TAATAATAATAATAA-3’Female 5’-TAATAATAATAATAA-3’

One proposed explanation for their fast rate of evolution is slippage during DNA replication.

Strand slippage replication at STR

The number of repeat units varies between alleles while the PCR primers bind to conserved flanking regions

7 repeats

8 repeats

Homozygote = both alleles are the same lengthHeterozygote = alleles differ and are resolved from one another

Microsatellites (STR)16 or more loci can be amplified at onceSensitivities to levels less than 1 ng of DNAAbility to handle mixtures and degraded samplesDifferent fluorescent dyes used to distinguish STR loci with overlapping allele size ranges

STR Multiplexing

DNA profilingDNA Evidence Can Exonerate As Well As Implicate

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Single nucleotide polymorphisms (SNPs)

Single base pair differences occur about once every 500-1000 bp.In most populations there is a common SNP, and several less common SNPs.About 3 million SNPs occur in the human genome, and these are becoming popular genetic markers.SNPs can be used just like other genotyping markers, but more loci typically must be used because only 4 alleles (G, G, C, T) are possible.

Typing a SNP PCR ApplicationsMolecular identification


Most mutations involve single nucleotide polymorphisms (SNPs)PCR amplifications with one primer binding to site of SNPOnly properly matching primer gives product

Use one primer/reaction for each known allele

Mutation detection Mutation detection

Huntington’s disease is an example of a microsatellite triplet repeat in a coding region

autosomal-dominant

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Prenatal Diagnosis of Genetic Diseases

Cells from amniocentesis

Possible carriers by buccal swab

Analyze relevant loci

644 bp440 bp

204 bp

Molecular analysis of a family with an autosomal recessive disease


Bioinformatics Mammalian/human genomesancestral genomes (Neanderthal)biomolecular computingTaxonomic relationships/phylogenies

Biomolecular computing


Bioinformatics Mammalian/human genomesancestral genomes (Neanderthal)biomolecular computingTaxonomic relationships/phylogenies

Taxonomicrelationships/phylogenies

Krause et al 2005

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PCR ApplicationsGenetic engineering

Molecular cloningSite-directed mutagenisisGene expression studies

Summary

blood, chorionic villus, amniotic fluid, semen, hair root, saliva, microorganisms, fossils, …

Gel Analysis, Restriction Digestion, Sequencing, Cloning

The speed and ease of use, sensitivity, specificityand

robustness of PCR has revolutionized molecular biology

and made PCR the most widely used and powerful

technique with great spectrum of research and

diagnostic applications.

Conclusion Quick Quiz

The purpose of PCR is to:

A. make copies of a specific region of DNAB. identify the presence of particular genesC. make sufficient genetic material for future

molecular workD. all of the above

Quick Quiz

A thermocycler protocol is comprised of which series of steps?

A. Decontamination, amplification, excisionB. Annealing, polymerization, coolingC. Transcription, translation, expressionD. Denaturing, annealing, extension

Quick Quiz

PCR amplifies molecular products in a(n) __________ fashion:

A. LogarithmicB. ExponentialC. LinearD. Random

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Quick Quiz

Primers:

A. Stabilize double stranded DNAB. Are enzymes that catalyze the copying

processC. Are short single-stranded DNA fragmentsD. Are long strings of nucleotides

Quick Quiz

Which of the following reagents is NOT in a master mix?

A. MgCl2B. Template DNAC. H2OD. dNTPs

Quick Quiz

If you forgot to add one of your primers your resultant gel will probably have

A. No bandsB. A smearC. A band of the wrong sizeD. Many bands

what is pcr? pcr

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