MAKERERE UNIVERSITY.

COLLEGE OF HEALTH SCIENCES.

SCHOOL OF BIOMEDICAL SCIENCES.
SPECIAL-DIAGNOCTIC TECHNIQUES. (SPECIAL STAINS AND
IMMUNO-STAINS)-CYT 2209.

ASSIGNMENT BY DR. NASINGHE EMMANUEL.

OWOMUGISHA ASUMPTA. 22/U/3785/PS.
MUWANGA SUUDI. 22/U/22608
MUGOMBA JULIUS. 22/U/6340
OKWIR JOSHUA NEWTON. 22/U/22875.

QN1; DESCRIBE REAL TIME PCR AND CONVENTIONAL PCR

Real time PCR. (QPCR)

 In real time PCR, the accumulation of amplification product is measured as the
reaction progresses in real time with product quantification after each cycle.
 Its workflow below delineates the steps in real time PCR. First, amplication reactions
are set up with PCR reagents and unique primers.
 Reactions are then run in real -time PCR instruments and the collected data is
analyzed by propriety instrument software.
 Real-time detection of PCR products is enabled by the inclusion of a fluorescent
reporter molecule in each reaction well that yields increased fluorescent with an
increasing amount of product DNA.
 The fluorescence chemistries employed for this purpose include DNA binding dyes
and fluorescently labeled sequence-specific primers or probes. Specialized thermal
cyclers equipped with fluorescence detection modules are used to monitor the
fluorescence signal as amplification occurs.
 The measured fluorescence is proportional to the total amount of amplicon. The
change in fluorescence over time is used to calculate the amount of amplicon
produced in each cycle.
 The main advantage of real-time PCR is that it allows you to determine the initial
number of copies of template DNA with accuracy and high sensitivity over a wide
dynamic range.
 Real-time PCR results can either be quantitative (presence or absence of a sequence)
or qualitative.

 Applications of real-time PCR include;

 Real-time PCR assays have become the tool of choice for the
rapid and sensitive determination and quantitation of nucleic
acid in various biological samples.
  In research laboratories, qPCR assays are widely used for the
quantitative measurement of gene copy number or the presence
of mutant gene.

 Two common methods for the detection of PCR products in real time PCR are non-
specific fluorescent dyes that intercalate with any double stranded DNA and sequence
specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent
reporter which permits detection only after hybridization of the probe with its
complementary sequence.

 Conventional PCR.
 This is a standard PCR process where the primers bind with DNA strands. Here, the
primers bind specifically to each other with to DNA strands. The primers used in PCR
are specific to the target sequence limiting the replication to that particular DNA
sequence. The process can be performed in PCR tubes containing aluminum blocks,
DNA polymerase, buffer, target DNA and primers. It takes about 35-40 minutes to
complete and is monitored using gel electrophoresis. The result of conventional PCR
are the identical copies of the target DNA sequence used for a variety of applications
including genetic research, medical diagnosis and forensic science.

o QN.2; DIFFERENTIATE BETWEEN REAL-TIME PCR AND

CONVENTIONAL PCR.

 Coventional PCR.  Real-time PCR.

 It is semi-quantitative.  It is both quantitative and
qualitative.
 It is time consuming and requires  It saves time.
manual manipulation
 It is cheap  It is expensive
 It has a low sensitivity  It has a higher sensitivity.
 Does not use fluorescent probes.  Uses fluorescent probes.