BABS1201 Lecture Notes - Lecture 13: Fluorescence, Anna Nicole Smith, Dna Paternity Testing
28 May 2018
School
Department
Course
Professor
Manipulating the Genome: PCR and individual variation
The Human Genome Project
• Completed in 2003
• To get the entire genetic sequence in a human
o Sequence a male, to get information in the Y chromosome
• Sequenced James D. Watson (Watson and Crick - DNA structure) and J. Craig Venter
(entrepreneur scientist - made it faster approach to sequence the DNA)
Genome - the entire genetic information contained within each individual cell
Analysing specific DNA sequences
• A single gene in the human is 1/1000 000th of the DNA
• Must detect the gene or viral DNA in the presence of billions of bases in the human DNA
o Uses PCR
Polymerase Chain Reaction
Requires the activity of:
• DNA polymerase
o Requires:
▪ DNA template - a single-stranded DNA
▪ Primer - short piece of DNA to add on to
▪ Free nucleotides - to add to the growing chain
•
• Primer
o Made so that it will code for the target sequence in the DNA
o Complementary to a section of the DNA
• DNA polymerase
o Adds GTP, ATP, CTP, TTP nucleotides to the DNA sequence
o Works at 37 degrees
o Synthesises a new DNA strand
• Separating strands
o DNA is heated to 100 degrees to break the hydrogen bonds between the two strands
o Separates two stands of DNA
find more resources at oneclass.com
find more resources at oneclass.com
PCR
•
• Target sequence - contains the sequence that we want to examine
• Primer - is synthesised to the two strands
o Complementary and identical DNA segments are made to attach to both strands
o Design primers that will bind to the top and bottom strands
• DNA polymerase - synthesises a new stand of DNA
• Heated to separate the two stands
Cycles of DNA amplification
Exponential amplification
Each cycle doubles the number of DNA molecules
1 copy is duplicated in each cycle, making 2� copies of the DNA
Original concept for PCR
Heat to 95 degrees to split the DNA strands
Cool it down to allow the primers to bind (anneal)
DNA polymerase synthesises the new strand at 37 degrees
This is repeated for the next cycle
o However, heating to 100 degrees will denature the DNA polymerase, so new DNA
polymerase will need to be added after each cycle
find more resources at oneclass.com
find more resources at oneclass.com
Taq polymerase
DNA polymerase from the thermophile Thermus aquaticus
o Stable at 98 degrees, works at 70 degrees
How PCR is performed now
Denature - DNA strands separate (95)
Anneal - Primers bind to DNA (55)
Extend - DNA polymerase synthesises new strand (72)
Automated PCR machine
Conducts PCR
Can hold 96 - 382 samples
Makes lots target DNA, billions of target sequence to 60 larger sequences, almost pure target
DNA is obtained
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
The human genome project: completed in 2003, to get the entire genetic sequence in a human, sequence a male, to get information in the y chromosome. Sequenced james d. watson (watson and crick - dna structure) and j. craig venter (entrepreneur scientist - made it faster approach to sequence the dna) Genome - the entire genetic information contained within each individual cell. Analysing specific dna sequences: a single gene in the human is 1/1000 000th of the dna, must detect the gene or viral dna in the presence of billions of bases in the human dna, uses pcr. Requires the activity of: dna polymerase, requires, dna template - a single-stranded dna, primer - short piece of dna to add on to. Separating strands: dna is heated to 100 degrees to break the hydrogen bonds between the two strands, separates two stands of dna. Each cycle doubles the number of dna molecules.
