Transcript PCR Polymerase Chain Reaction

PCR
Polymerase Chain
Reaction
Mariam Cortes Tormo
Miami Children’s Hospital
Research institute
2013
Amplification reaction
Polymerase Chain Reaction (PCR) is done in three steps
that constitute a cycle, repeated for a certain amount
of times:
1 - Denaturation
2 - Hybridization
3 - Elongation
The time, temperature and number of cycles are factors
that determin the results of the PCR, thus by modifying
we can optimize the reaction
Denaturation
This is a critical step as it is very important that the DNA
template be completely denatured
•
•
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It is recommended temperatures of 94°-95° C, for 30'' to1‘
High G. + C increases the time or temperature
The enzyme activity decreases very rapidly to from 95 ° C. At
these temperatures or higher is recommended to decrease the
incubation time
In practice, before start
cycles, is normal to add
a denaturation period
This step is usually
94°C 5'
Hybridization
Temperature and time will depend on 3 factors related to the founders:
• Composition of bases
• Size
• Concentration
In practice, the annealing
temperature may range between
45°C and 65°C, for a time
ranging from 30” to 1’
An increase in temperature
promotes the specificity; where
as a decrease will promote
incorrect connections of the
initiators
Increased time promotes less
specificity for
nonspecific amplification
products
Elongation
In most reactions, the extension step performed at 72 ° C.
This temperature can range between 70-72°C
The extension time depends on the size of the amplification. You
can estimate about 1 minute to 1 kb elongated
In practice it is normal for the end of all cycles to perform an
ultimate elongation of 72°C from 5'
Amplified DNA
Cycles
Some things to know…
...to get the best conditions to reduce errors:

Not using a high number of cycles (25-45). The error
rate is proportional to the number of cycles

The concentration of dNTPs must be equal for the 4
types, while being as low as possible without losing
performance
Between 20-200 mMol
Reduce the time as much as possible in each step
The concentration of Mg ++ should not be excessive.
This decreases the specificity of PCR
The concentration of Mg ++ must be 0.5 - 1.0 mM
times the concentration of dNTPs



Dimethyl sulfoxide (DMSO 10%) contributes to
decreasing the secondary structure of DNA

Buffers which help to stabilize the enzyme
How many PCR’ s can we
find?
Nested PCR
 In situ PCR
 Multiplex PCR
 RT-PCR
 Real Time PCR
 Random primer PCR

Nested PCR
Highly specific

Nested primers are used to increase the specificity of an amplification or to
reamplify the product of a PCR reaction that did not yield enough material

Purpuse : Diagnosis of infectious diseases
In situ PCR
Mainly used in two aspects
•
Detection of exogenous gene fragment, improve the detection rate of virus infection,
concentrated in the inspection, such as HIV, HPV, HBV, CMV, etc
•
To observe the distribution of pathogens in the body
•
Endogenous gene fragments, such as human monogenic disease, gene recombination,
translocation of chromosomes, Ig fragments of mRNA gene fragments, etc
•
Detection of gene transfection
Multiplex PCR
Multiplex polymerase chain reaction (PCR) is a variant of PCR in which
two or more loci are simultaneously amplified in the same reaction
Since its first description in 1988, this method has been successfully
applied in many areas of DNA testing, including analyses of deletions,
mutations and polymorphisms or quantitative assays and
reversetranscription PCR
Some of the applications of multiplex
PCR include:
•
Pathogen Identification
•
High Throughput SNP Genotyping
•
Mutation Analysis
•
Gene Deletion Analysis
•
Template Quantitation
•
Linkage Analysis
•
RNA Detection
•
Forensic Studies
•
Diet Analysis
RT-PCR
This technique is commonly used in molecular biology to detect
RNA expression levels
It is used to qualitatively detect gene expression through creation
of complementary DNA (cDNA) transcripts from RNA
• Genetic Disease Diagnosis :RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan
syndrome. This genetic disease is caused by a malfunction in the HPRT1 gene, which clinically leads to the fatal
uric acid urinary stone and symptoms similar to gout. Analyzing a pregnant mother and a fetus for mRNA
expression levels of HPRT1 will reveal if the mother is a carrier and if the fetus will likely to develop Lesch–
Nyhan syndrome
• Cancer Detection : Scientists are working on ways to use RT-PCR in cancer detection to help improve
prognosis, and monitor response to therapy. Circulating tumor cells produce unique mRNA transcripts depending
on the type of cancer.
• RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such
as Influenzavirus A and retroviruses like HIV
• RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes
Real Time PCR
Is based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously
quantify a targeted DNA molecule
Random primer PCR
TECHNIQUE USED PRIMES CONSISTING OF A POOL OF SEQUENCE
ENDS 3 'REGION OF DNA AND A 5' CONSTANT FOR THE DETECTION
OF FIRST BUILT
APPLICATION ALLOWS THE AMPLIFICATION OF DNA MOLECULES
AND GENOME THAT VARY FROM 400 TO 40 PAIRS OF MEGA BASES
THIS VERSION OF TECHNICAL SPECIFICATIONS IS VERY SENSITIVE
AND IS VERY USEFUL IN THE STUDIO AND DIAGNOSIS OF BODIES
OF THE SAME KIND WITH SPECIFIC GENETIC VARIATIONS
Thank you
very much
for your
attention