In this slides the topic that which is discussed is "How PCR is involved in identification of Genotype" I hope this will Help you in your presentation work. "PCR can be used in identification of genotype."

1. PCR In Identifying
GenotypesPresented to -
Dr. Hasan Mahmud Reza
Course: Biotechnology
Course code: PHR 325
Section : 03
North South University
Department of Pharmaceutical Science

2. Group Members
• Sarah Bin Sultana
• Jobeda Khanom Lina
• Shamim Hossen
• Md. Naimul Islam
• Stanley Lawrence Palma

3. History of PCR (Polymerase chain reaction)
•The polymerase chain reaction (PCR)
was originally developed in 1983 by
the American biochemist Kary Mullis.
He was awarded the Nobel Prize in
Chemistry in 1993 for his pioneering
work.
•PCR is used in molecular biology to
make many copies of (amplify) small
sections of DNA or a gene.

4. What is PCR?
•PCR is a common tool used in medical and biological
research labs. It is used in the early stages of processing
DNA for sequencing, for detecting the presence or
absence of a gene to help identify pathogens during
infection, and when generating forensic DNA profiles
from tiny samples of DNA.
•Using PCR it is possible to generate thousands to
millions of copies of a particular section of DNA from a
very small amount of DNA.

5. Principle of PCR
•PCR principle: It is a chain
reaction: One DNA molecule is
used to produce two copies,
then four, then eight and so
forth. This continuous doubling
is accomplished by specific
enzymes known as
polymerases, that are able to
string together nucleotides to
form long molecular DNA
strands.

6. What is Genotype
• The genotype of an organism is the chemical
composition of its DNA, which gives rise to the
phenotype, or observable traits of an organism. A
genotype consists of all the nucleic acids present in a
DNA molecule that code for a particular trait. The
outward appearance, or phenotype, is the result of
interactions of proteins being created by the DNA.
• Modern DNA analyzing techniques have made it easier
to identify which segments of DNA are responsible for
various phenotypes.

7. Example of Genotype
• Although a particular genotype consists of many nucleic acids,
scientists typically represent genotypes with single letters, or two
letters in the case of sexually reproducing organisms that receive one
allele from each parent.
• For example, the trait for eye color could be represented with the letter
“E”. Varieties, or alleles, of that trait that are dominant will be
designated by capital letters. Therefore, “E” will represent brown eyes.
Traits that are recessive are written in lower case. The allele for blue
eyes is recessive to the allele for brown eyes, so we can call it “e”.

8. Example of Genotype
• So there could be 3 genotypes for this eye colour
1) Homozygous Dominant “EE” “EE” =
2) Heterozygous Dominant “Ee” “Ee” =
3) Homozygous Recessive “ee” “ee” =
Here,
E = Brown eye color
e = Blue eye color

9. Genotypes consist of nucleotide. In PCR
process we can detect variation of DNA
sequences .
In this picture we can see that it has single
nucleotide polymorphism(SNP).
Here provide sequence-specific primer(SSP)
Single stand DNA(ssDNA)
If provide sequence-specific primer-1(SSP-1)
Primer extension is occurred.
Exact match
SNP identify: G
Genotyping by PCR

10. Genotyping by PCR
If provide sequence- specific primer-2(SSP-2)
Primer extension is not occurred.
Mismatch
SNP identify : Unknown
After completing the PCR for detecting
genotypes,
Comparer the length of products using gel
electrophoresis

11. Restriction Fragment Length Polymorphisms (RFLP) Analysis
Typical RFLP analysis comprises five major
steps
1.DNA isolation and purification
2.PCR amplification and restriction enzyme
digestion
3.Separation and detection of the digested
products via electrophoresis
4.Analysis of data to generate the fragment
profile for each sample
5.Clustering analysis based on the profile of
samples from step 4

12. Steps of Mouse Genotyping
Know What you are Looking For
If we have detailed information about the genomic modification, we
can identify the gene sequence under investigation.
Pick the Correct Technique for your Mouse Genotyping
For all further characterizations of offspring from established mouse
lines, use PCR, because it’s quick and easy. If we need higher
sensitivity and reliability, use a quantitative PCR with a melting curve
analysis

13. Steps of Mouse Genotyping
Design the Right Primers
• The most important component of any PCR reaction is a perfect set
of primers. Spend the most time and effort at this step.
• If our mice came from a trusted source with a set of genotyping
primers, then great. we’re all ready to test them in a PCR. If not,
design primer sets like you would for a regular PCR reaction.
Material for DNA Isolation
• There are several ways to obtain DNA for mouse genotyping. The
one we use depends on the established practice in your lab and
the quantity of DNA required for the assay.
• we can collect DNA from ear tissue (0.5 to 2 mm) using an ear
punch

14. Isolating Genomic DNA for Genotyping
Once we have our sample tissue in hand, it is time to isolate genomic DNA from the
cells.
PCR
Standardize your genotyping PCR the same way we would for any PCR. Some points
to consider:
• Optimize your PCR primers using positive controls
• Titrate the amount of genomic DNA to find the optimum template concentration
• Always include wild type and mutant control samples to compare bands on gel or
curves in a melt curve analysis
• Often, the difference in the band sizes of the wild type and mutant bands will be
very small – make sure you get good separation in the gel by using a longer run

15. Rapid PCR-Based to Determine Genotype
of Lactococcus lactis Subspecies
• A highly efficient, rapid, and reliable PCR-based method for
distinguishing Lactococcus lactis subspecies (L.
lactis subsp. lactis and L. lactis subsp. cremoris).
• Lactococcal strains are essential to milk fermentation, especially
in the cheese-making process, providing optimal conditions for
curd formation and for the development of texture and flavor.
• Isolates from cheese starters were investigated by this method,
and amplified fragments of genetic variants were found to be
approximately 40 bp shorter than the typical L.
lactis subsp. cremoris fragments.

16. Materials and methods
• Recently, a novel criterion for distinguishing L.
lactis subsp. lactis from L. lactis subsp. Cremoris has been reported
is glutamate decarboxylase (GAD) activity, which has been
observed in L. lactis subsp. lactis and not in L.
lactis subsp. Cremoris.
• The gadB gene encoding L. lactis subsp. cremoris GAD was
apparently inactivated by a frameshift mutation resulting from an
adenine deletion or a thymine insertion and encoded a
nonfunctional protein.
• The gadB fragments were amplified by PCR, using the totalL.
lactis DNA or the first-strand cDNA as the template.

17. Materials and methods
• The PCR primers were designed from the published sequence
of L. lactis gadB. The sense primer (gadB21) was located within
the gadB gene, and the antisense primer (GAD7) was located
downstream from the gene.
• Each 50 μl of PCR mixture contained 200 ng of genomic DNA,
20 pmol of each primer, reagent mixture, and Ampli Taq gold
DNA polymerase . PCR amplification was conducted with a
GeneAmp PCR System 2400
• The PCR conditions were as follows: denaturation at 94°C for 9
min, followed by 45 cycles of denaturation at 94°C for 30 s,
annealing at 50°C for 30 s, and extension at 72°C for 60 s, with
an additional extension of 7 min at 72°C after the last cycle.

18. Conclusion
• The polymerase chain reaction is a very helpful way
to replicate DNA ,diagnosis certain diseases and
identification of genotype. It is very reliable way to
test and analyze the DNA and is also very efficient in
the sense that is only take a few hours .

19. References:
• https://www.the-scientist.com/lab-tools-old/genotyping-with-pcr-46437
• https://biologydictionary.net/genotype/
• https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-
reaction
• https://aem.asm.org/content/68/5/2209
• https://aem.asm.org/content/66/5/2235?ijkey=438f2836744a06a982e3d4
11eda9c17b3778bcd2&keytype2=tf_ipsecsha
• https://www.youtube.com/watch?v=urmkEf5iL6I&feature=share&fbclid=Iw
AR0MuB3flXRHZcSHW6YTJda2yB7D0xvFJf0AANLlVZl5nFgYY2D1bP2N05Y