Sie sind auf Seite 1von 118

PCR and its application

Continued..

Ref: Cloning Heterologous Genes: Problems and Approaches

Jacqueline Agnan, Christopher Korch and Claude Selitrennikoff


Fungal Genetics and Biology 21, 292301 (1997) ,Article No. FG970995 Wikipedia and cross references

Thermostable Polymerases
Taq: Thermus aquaticus (most commonly used)
Sequenase: T. aquaticus YT-1 Restorase (Taq + repair enzyme)

Tfl: T. flavus Tth: T. thermophilus HB-8 Tli: Thermococcus litoralis Carboysothermus hydrenoformans P. kodakaraensis (Thermococcus) Pfu: Pyrococcus furiosus

Thermostable Polymerases
Polymerase Taq pol Amplitaq (Stoffel fragment) Vent Deep Vent Pfu Tth T , 95oC 40 min 80 min Extension Type of Rate (nt/sec) ends 75 3A >50 3A Source T. aquaticus T. aquaticus

400 min 1380 min >120 min 20 min

>80 ? 60 >33

95% blunt 95% blunt Blunt 3A

Thermococcus litoralis Pyrococcus GB-D Pyrococcus furiosus T. thermophilus

APPLICATION

APPLICATION OF PCR &

VARIENTS OF PCR

Analysis of PCR Products


Analysis of Amplicons
Restriction Digestion Electrophoresis

Cloning Electrophoresis DNA Sequence

Southern Electrophoresis
Fragment Size Transfer

Amplicon
Size

Analysis

Hybridize
with Probe

Detection

Application in Genetic Engineering

Gene cloning
Cloning by PCR, DNA is amplified to analyze it
Cloning by plasmid followed by transformation, DNA is stored for long time. Bacteria divide and reproduce, the plasmid gets copied and therefore, so does the DNA fragment

Cloning - PCR Strategy


The gene of interest usually has to be amplified from genomic or vector DNA by before it can be cloned into an expression vector. The first step is the design of the necessary primers Not to have: 3'-end : 3 of more G or C bases at this position (this may stabilize nonspecific annealing of the primer); a 3' thymidine (it is more prone to mispriming than the other nucleotides). Primer pairs should be checked for complementarity at the 3'-end. This often leads to primer-dimer formation. Bases at the 5'-end of the primer are less critical for primer annealing. Therefore, it is possible to add sequence elements, like restriction sites, to the 5'-end of the primer molecule. Primer length, GC content, Tm

Cloning Heterologous Genes


Many economically and medically important fungi are genetically less well characterized, which makes it difficult to study their genes and gene products. Cloned genes from model organisms are often used to identify and isolate the cognate genes from these fungi on the basis of assumed conservation of sequence and function

Heterologous probes may not always detect a cognate gene


The availability databases its possible to identify conserved regions of genes, which can be the basis for PCR amplification and cloning of a genomic or cDNA segment of a cognate gene of unknown sequence The amplified segment can subsequently be used as a homologous probe to screen a DNA library

Obtaining a homologous product by PCR is not always simple.


Successful amplification of a DNA segment of unknown sequence by PCR depends primarily on the sequence of the primers. Designing primers that amplify a specific DNA sequence from a heterologous organism can be a challenging task. Amplification of a DNA segment of unknown sequence can be done by using oligonucleotides of either specific or degenerate sequence If the model gene has been sequenced in-house, it is worthwhile to try the sequencing primers first.

Degenerate primers
These are actually mixtures of similar, but not identical primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical The other use for degenerate primers is when primer design is based on protein sequence, as several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case Isoleucine might be "ATH", where H for adenine, thymine, or cytosine.

Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR

TOUCH DOWN PCR


(Step-down PCR):
A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles

Overlap extension polymerase chain reaction

For splicing Deleting a portion Induction of mutation

Splicing by Overlap Extension PCR

Joining two DNA sequences

Deleting a DNA sequences

DNA Ploymorphism

Amplicon size
For genetic diversity/ relation

For identification of individual


Marker

Genetic fidelity

DNA Polymorphism

Some of the conventional PCR based markers RAPD - dominant AFLP - dominant

ISSR - dominant
SSR co-dominant

Genetic fidelity

Allele-specific (AS-) PCR:


A diagnostic or cloning technique based on single-nucleotide polymorphisms (SNPs) .

It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP.

PCR Amplification of Specific Alleles (PASA)


Forward for G allele: 5-CCTGGACAACATGGTGACACTCC-3

Forward for C allele: 5-CCTGGACAACATGGTGACACTCG-3


Reverse common: 5-GTGGGCCCTGTACTTTTACATC-3

DNA Fingerprinting
Identification of individual
Criminal identification Forensic Sc. Parentage Plant/Animal/Species identification Seed material etc.

Diagnostic PCR Amplification From Patient Samples

104 bp

Multiplex-PCR
Consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. A single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized

Asymmetric PCR
Preferentially amplifies one DNA strand in a double-stranded DNA template It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required

DNA Sequencing

Chain Termination (Sanger) Sequencing


A modified DNA replication reaction. Growing chains are terminated by dideoxy nucleotides.

Chain Termination (Sanger) Sequencing


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

Chain terminates at ddG

Chain Termination (Sanger) Sequencing


A sequencing reaction mix includes labeled primer and template.
Primer

5OPTemplate

-3 OH TCGACGGGC
Template area to be sequenced

Dideoxy nucleotides are added separately to each of the four tubes.

Chain Termination (Sanger) Sequencing


A

ddATP + four dNTPs


ddCTP + four dNTPs

ddA dAdGdCdTdGdCdCdCdG
dAdGddC dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC dAddG dAdGdCdTddG dAdGdCdTdGdCdCdCddG

ddGTP + four dNTPs

ddTTP + four dNTPs

dAdGdCddT dAdGdCdTdGdCdCdCdG

Chain Termination (Sanger) Sequencing


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.

Chain Termination (Sanger) Sequencing


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:

Classical Sequencing Gel

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 multi-cyclic 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 dyeconjugated 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 dideoxy nucleotides, labeling the sequencing ladder at the 3 ends of the chains.

ddA

ddA

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 AC G T GT

The fragments are distinguished by size and color.

AT A GLANCE

Sequencing:

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)


Steps in the RT-PCR reaction:
RNA isolation Reverse transcription PCR amplification Analysis of PCR product

Reverse Transcription of RNA (RT)


Primer Options for RT reaction Oligo (dT) Random Hexamers Sequence-specific Primers Enzyme Options for RT Reaction Retroviral RNA-directed DNA polymerase AMV Reverse Transcriptase (avian myeloblastosis virus) MMLV Reverse Transcriptase (Moloney murine leukemia virus)

Real Time PCR (RT-PCR)


qPCR
A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer).

Fluorescent reporter probe method

LATE-PCR
A recent modification on this process, known as Linear-After-TheExponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction

Smiths Detection has an exclusive license for LATE-PCR from Brandeis University for all markets, worldwide This enables one to detect low numbers of target organisms

Assembly PCR / Polymerase cycling assembly ( PCA)


A method for the assembly of large DNA oligonucleotides from shorter fragments.
The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process. It thus allows for the production of synthetic genes and even entire synthetic genomes.

The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product

Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR

Isolation of a gene by PCR If the Primers anneals anneal both the sides of the gene of interest No selection required Then why cloning Limitation: Sequence information ids required Length of DNA sequence If seq information is not known

If we have information on heterologous gene / equivalent gene

Its important to isolate gene whose seq is known

Fish out gene - based on flanking sequence information

Allelic forms of genes can be isolated

Diagnostics both in plant and animal including human

Early diagnosis

Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from nonhomologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct.

Cloning
Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule.
These cells are clones, hence the name

This used to be the only way to amplify DNA. It is still by far the most accurate.

Plasmid vectors circular, autonomous bacterial DNA

The vector is made with a T overhang

Taq polymerase leaves an A overhang


Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR.

When Taq synthesizes a new strand, it always puts an extra A at the end
This can be useful, but note: other polymerases do not do this, they leave blunt ends. Only Taq polymerase leaves A overhangs. Blunt end vectors do not work with Taq, we need a T overhang.

DNA ligase
Repairs gaps in the sugar-phosphate backbone of DNA
Creates phosphodiester bonds

Does not do anything with the bases

Transformation of bacteria
Two main methods for transformation
Chemical / Heat Shock As done in last practical, this method gets DNA into the cell by making them porous using CaCl2 and a 42 C heat treatment Electroporation Makes cells porous using high-voltage electricity

Imperfect science
Most of the plasmid / insert combinations will not ligate
Most of the bacteria will not be transformed

We only need one molecule to get into one bacterium to make one colony.

PCR from clones


Often clones will religate containing any old DNA (eg primer dimers)..
The DNA can go in in either orientation

We can use the PCR to tell which colonies have the insert we want, and which orientation it is in.

Touch down PCR


Touchdown PCR involves decreasing the annealling temperature by 1 degree C every second cycle to a 'touchdown' annealing temp which is then used for 10 or so cycles

It was originally intended to bypass more complicated optimization processes for determining optimal annealing temperatures.

The idea is that any differences in Tm between correct and incorrect annealing gives a 2-fold difference in product amount per cycle (4fold per degree C). You therefore enrich for the correct product over any incorrect products.

DD-PCR
To detect the differential expression of the gene Usually subtractive-hybridization and differential cDNA cloning
require large amounts of mRNA derived from cell or tissue samples, and are very laborious and time consuming, often taking many months from start to finish.

Alternatively

Differential Display PCR: requires only small


amounts of cell or tissue samples, and can be completed within weeks.

Requires RNA derived from control (normal) and sample (diseased) cells or tissues

The samples of RNA being examined are converted into cDNA by reverse transcription using an oligo-dT primer with two penultimate specific bases to the 3 end 5 TTTTTTTTTTTTMN 3 (where M = A, G, or C; N = any nucleotide)

Upon conversion to cDNA the PCR amplification is done with arbitrary 5 primer (8-10mers) using random sequences, and the same 3 primer (shown above)

In order to define most of the genes expressed in these cells, different sets of random primers may be used

After ~30 amplification cycles using radioactive S-35 labeled nucleotides the expressed cDNA fragments are displayed using DNA sequencing gels

Differentially expressed cDNAs are easily visualized following autoradiography


The DNA from these gels slices is recovered by boiling, and reamplified by PCR using the original primers. The amplified DNAs are characterized by sub-cloning, sequencing and data-base searches

To verify the differential expression status of each of these sub-clones, Northern-Blot assays are performed using RNA isolated from the original sample sources

COLONY PCR
Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from nonhomologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement

MULTIPLEX PCR
Two or more sets of primers, designed for amplification of multiple target
Multiple targets can be detected from a single specimen in one reaction More complicated and less sensetive

TRANSCRIPTION MEDIATED AMPLIFICATION OR NUCLEIC ACID SEQUENCE BASED AMPLIFICATION


Isothermal amplification techniques RNA to DNA and DNA as a template for multiple copies of RNA (Like the life cycle of retrovirus)

TMA/ NASBA

Detection of HIV, HCV, vericella zoster virus, cytomegalovirus, rhinivirus, measles, papillomavirus, M. tuberculosis, M. pneumoniae etc.

STRAND DISPLACEMENT AMPLIFICATION

Isothermal amplification technique


To detect trace amount od DNA or RNA of a particular sequence Detection of tuberculosis, trachomatis, gonorrhoea

STRAND DISPLACEMENT AMPLIFICATION

SDA

LIGASE CHAIN REACTION


Two oligonucleotide probe hybridise side by side A thermostable DNA ligase seal the nick Each ligated product as well as original target serve as a template A modification of this technique gapped LCR (G-LCR)
Kits are available for the detection of C.trachomatis, N. gonorrhoeae etc.

LCR

SIMPLE METHOD TO SEQUENCE LAMBDA, M13, OR PLASMID CLONES USING PCR


Three major steps
1. Denaturation at 94C

2. Annealing at 50C

1. extension at 60C

Detection

SEQUENCING

REFERENCE BOOKS
Dieffenbach, C.W and Dvksler, G.S. (1995) PCR primer: a laboratory manual. CSHL press, Cold Spring Harbor, USA.

Erlich, H. A. 1989. PCR technology: Principles and applications for DNA amplification. Stockton Press, New York.

Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990. PCR Protocols: A guide to methods and applications. Academic Press, New York.

Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing. Eaton Publishing Co., Natick, Mass.

Bej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology 26:301-334.

Denature (heat to 95oC)

Lower temperature to 56oC Anneal with primers

Increase temperature to 72oC DNA polymerase + dNTPs

DNA copies vs Cycle number


2500000

2000000

DNA copies

1500000

1000000

500000

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cycle number

Applications of PCR
Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis

Applications of PCR
Basic Research
Mutation screening Drug discovery Classification of organisms Genotyping Molecular Archaeology Molecular Epidemiology Molecular Ecology Bioinformatics Genomic cloning Site-directed mutagenesis Gene expression studies

Applied Research
Genetic matching Detection of pathogens Pre-natal diagnosis DNA fingerprinting Gene therapy

Applications of PCR
Molecular Identification
Molecular Archaeology

Sequencing
Bioinformatics Genomic cloning Human Genome Project

Genetic Engineering
Site-directed mutagenesis

Molecular Epidemiology
Molecular Ecology DNA fingerprinting Classification of organisms

Gene expression studies

Genotyping
Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens

MOLECULAR IDENTIFICATION:

Molecular Identification:

Detection of Unknown Mutations

SSCP gels: shifts representing a mutation in the amplified DNA fragment

Molecular Identification:

Classification of Organisms
1) Relating to each other * Fossils
2) Similarities 3) Differences * Trace amounts * Small organisms Insufficient data

! DNA !

Rademaker et al. 2001

Molecular Identification:

Detection Of Pathogens

Molecular Identification:

Detection Of Pathogens

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

Molecular Identification:

Genotyping by STR markers

Molecular Identification:

Prenatal Diagnosis
Chorionic Villus
Amniotic Fluid

644 bp 440 bp 204 bp

Molecular analysis of a family with an autosomal recessive disease.

DNA sequencing Sangers method

SEQUENCING
Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide!

Fragments of DNA differing only by one nucleotide are generated

Nucleotides are either

or

Sequencing:

Reading Classical Sequencing Gels

Summary

blood, chorionic villus, amniotic fluid, semen, hair root, saliva

68,719,476,736 copies

Gel Analysis, Restriction Digestion, Sequencing

Conclusion
The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.

Gene Cloning and DNA analysis: An Introduction 6th ed. T.A. Brown, Blackwell