Week 8 Flashcards
What are the PCR components and their role?
- DNA samples
- Primers: it is a short nucleic acid sequence that provides a starting point for DNA synthesis.
- Nucleotides: Nucleotides are the monomers of the polymer Nucleic Acids. It is the building blocks of DNA and RNA.
It is formed by a phosphate group, a sugar group and a nitrogenous group (Pyrimidines (cytosine-C, thymine-T, Uracil-U => single stranded) & Purines (Adenine-A, Guanine-G => 2 rings)). - Taq Polymerase: the role is to amplify the DNA for the production of multiple copies of DNA
- Mix buffer
- PCR tubes
What are the steps in PCR
- Denaturation: the temperature is increased to separate the double stranded DNA so we could have a template for our next experiment. The bonds that will be broken are found in between the double stranded DNA (between the complementary basis/nitrogenous bases) (94-96*C)
- Annealing: Temperature is decreased to allow primers to base pair to complementary DNA template (to give primer a chance to create a hydrogen bond, so that he can anyle (~68*C)
- Extension: Polymerase extends primer to form nascent DNA strands (72*C)
Name three exemples for PCR use
- Genetic Testing: screen for and detect DNA mutations (e.g. prenatal diagnosis)
- Genetic fingerprinting at crime scenes; paternity testing
- DNA sequencing (e.g. Sequencing of the human genome)
What is PCR?
The PCR technique allows amplification of a DNA sequence of interest.
Every time the cycle is performed, the number of copies of DNA sequence of interest is doubled.
It is use to discover new diseases and it simplify the DNA sequence.
Viruses
They are parasites. They use cell’s machinery to copy itself (reproduce)
Viruses cannot reproduce, the only way they can survive is by putting their DNA in our bacteria and the mRNA will reproduce them and so there will be more viruses.
How can prokaryotes defend themselves against viruses?
Prokaryotes have restriction enzymes that can cut DNA
Describe Restriction Enzymes (RE)
They are a defense mechanism in prokaryotes. They cleave (divide) foreign (strange/unfamiliar) viral DNA/RNA at specific sites.
Different restriction enzymes cleave different DNA/RNA patterns.
Most restriction sites are palindromes (DNA or RNA sequence that reads the same in both directions).
Cleaving may result in ‘’sticky’’ or ‘’blunt’’ ends.
What are the steps used in DNA Fingerprinting?
- DNA extraction
- PCR amplification (so that we can produce many copies of that specific sequence, so it will amplify the desire fragment)
- Restriction digestion (the DNA fingerprints will be cleaved by restriction enzymes) (it will distinguish between samples of the genetic material)
- Electrophoresis (the DNA sequence cleaved will be run through gel electrophoresis)
First your DNA is amplified using PCR, then cut into your restriction enzymes, then filtered to show your unique banding pattern with gel electrophoresis
How can the differences in a DNA sequence be detected and visualize?
By DNA fingerprinting
Ameligenin gene (AMEL)
It exists on both X and Y chromosomes and it has been used to determine the sex in cattle (animal) and human. It encodes a protein that is critical for the formation of the enamel on teeth.
AMELX gene provides instructions for making a protein called amelogenin, which is essential for the tooth development.
What is an Intron?
They are non-coding regions in a gene, eventually eliminated, during processing of the RNA transcription.
They are nucleotides in between exons that is not required in a mature RNA.
⭐️The introns of the AMELX and AMELY differ by 6 nucleotides!
How would we separate the two different copies of the amelogenin gene? (PCR used for the amelogenin gene)
Gel Electrophoresis
What is a plasmid?
Plasmids are another stream of nucleotide find in our bacteria. They are a circular or linear double-stranded DNA with it’s own origin of replication (every plasmid has its own). They are copied and transferred (vertically) to daughter cells during cell division.
There is horizontal transfer of plasmid (conjugation) between members of the same population/species and different populations/species (even different domains)
E.g.defense (antibiotic resistance), etc
What is Recombinant DNA technology?
It is a technology that uses enzymes to cut and paste together DNA sequences of interest.
It is used to identify, map and sequence gene, and to determine their function.
Exemple of Recombinant DNA technology
- Growth Hormone (go see power point 34 for graphic with steps)
- Production of Insulin (go see power point 35 for graphic with steps)
