Kornelia Mytsak
Shunkoh Nabeshima
Emily Shadbolt
Lisa Schumacher
PCRandDNAdiagnostics
DNA
• DNA contains loads of information neatly stacked and compressed to small sizes that can fit
into the cell nucleus.
• A single DNA molecule has two strands that wrap around one another to form a double helix.
• Every single strand of DNA is composed of the sequences of four nucleotides which the
individual letters were building blocks of DNA
• Nucleotides of DNA made up of sugar deoxyribose, phosphate, and one of the four nuclear
bases, adenine (A), cytosine (C), guanine (G), and thymine (T).
• The nucleotide of the one strands forms hydrogen bonds to complementary nucleotides and
other strands, specifically A bonds to T by two hydrogen bonds, and C bonds with G with three
hydrogen bonds.
• Two DNA strands also have a direction, which means one of them runs from 3’ end 5’ end while
the other runs from 5’ end to 3’ end.
• Every single protein of our body is encoded in combinations of just four nucleotides.
Polymerasechainreaction(PCR)
• PCR is its technique in molecular biology to amplifies a segmented DNA.
• To analyze it, we need lots and lots of copies of DNA to make it much easier.
• PCR is based on DNA replication, a process used to duplicate the genetic material
dividing into two identical daughter cells by using thermal cycling.
• DNA that we wish to multiply and enzyme Taq polymerase, primer, nucleotides (A, T, C,
and G).
• Thermal cycle of 96°C (denaturation), 55°C (annealing), 72°C (extension).
• The whole process lasts for 10 min.
• About an hour is enough to analyze with electrophoresis.
Gelelectrophoresis
• DNA is chopped up into smaller fragments using restriction enzymes, which are enzymes that
break the DNA at specific nucleotide sequences.
• Then the DNA fragments are poured into a well within a piece of agarose gel.
• An electrical current is passed through the gel and pulls the negatively charged DNA fragments
through the gel towards the positive end.
• Each band would represent a different size of DNA fragments.
• Use the PCR ladder to read the result.
• Do not forget to make a negative and positive control as well.
• Keep in mind that the restriction enzyme doesn’t always cut the nucleotide sequence that we
primarily intended to.
Restrictionfragmentslengthpolymorphism(RFLP)
• Depending on DNA sequence on an individual, cleaving off the chromosomal DNA
with restriction enzymes leads to DNA fragments of the variable lengths.
• The cleavage of DNA in specific sequences can be used as a polymorphic marker.
• These markers out of specific spots identify polymorphism using a restrictive
enzyme.
• The fragments are analyzed by Southern blot and detected by the probe.
Singlenucleotidepolymorphisms(SNPs)
• DNA sequence variants in a population that differ by only a single base pair.
• They are usually caused by errors during DNA replication and are point mutations.
• The difference can be seen from the number of bands on the gel electrophoresis,
which helps us to identify the gene mutations.
Microsatellites(shorttandemrepeats;SRTs)
• The repetitive sequence of several base pairs in DNA that are highly polymorphic.
• Belong to the variable number tandem repeats (VNTRs), which are short nucleotide
sequences in the genome of an individual that are repeated a variable number of
times.
• The common pattern of VNTRs in the repetition of trinucleotides for about 5~50
times or even more.
• DNA polymerase may copy more and changes the length of the allele.
• Then restriction enzyme added, and the fragments would be differed by showing up
using gel electrophoresis.
References
https://en.wikipedia.org/wiki/Polymerase_chain_reaction
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-
sequencing-pcr-electrophoresis/a/gel-electrophoresis
https://www.osmosis.org/
Lecture and practice presentation slides