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BCMB20005- Midterm Exam Guide - Comprehensive Notes for the exam ( 27 pages long!)


Department
Biochemistry and Molecular Biology
Course Code
BCMB20005
Professor
Amber Willems
Study Guide
Midterm

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UniMelb
BCMB20005
MIDTERM EXAM
STUDY GUIDE

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Afyeda BCMB20005 Notes Semester 1 2017
BCMB20005: TECHNIQUES IN MOLECULAR SCIENCE LECTURE NOTES
Lecture 4
All figures from Lecture 4 slides, A Willems-Jones, March 2017, Melbourne University (unless stated)
Need to Know for Exams
Techniques based on hybridisation
- PCR
o Principle, mechanism and applications
o Modern cloning using PCR compared to traditional cloning strategy
- RT-PCR
o cDNA: what is it and how is it generated?
o Process of RT-PCR
- Quantitative/Real time PCR
o Two types
o Principle
Polymerase Chain Reaction (PCR)
- Developed in 1983 revolutionised molecular biology and cloning
- PCR: in vitro amplification of a DNA sequence
o Exponential copying of few DNA molecules into thousands-millions of copies
- Utilizig eltig ad aealig popeties of DNA
- PCR relies on thermal cycling (i.e. heating and cooling of reaction)
o 3-steps: denaturing, annealing, extension
Double stranded molecule is separated into single strands
Hybridization of synthetic primers complementary to sequence of DNA.
o Need DNA that is thermostable: Taq
- Requires some knowledge of the sequence of the gene of interest for primers
o Sequence specific primers hybridize complementary DNA
- Provides an efficient alternative strategy to traditional cloning method.
o While southern blotting included the use of huge DNA piece and then finding the specific
sequence we are interested in
o PCR includes designing of primers (oligonucleotides of DNA) that contain specific restriction
eze sites ’ ed
Ligate digested amplified DNA into plasmid vector recombinant plasmid.
Applications of PCR
- Paternity testing
o Genetic markers
o Looking at variation between people
- Genetic testing for cancer predisposition
- Amplify rare DNA for evolutionary analyses
- Gene expression
- DNA cloning
- Test for disease causing pathogens
- Forensic analysis
- Human remains identification
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Afyeda BCMB20005 Notes Semester 1 2017
Requirements
- Template DNA
- DNA polease, e.g. Ta polease: atalse stepise additio of uleotides at ’ ed
o Requires a pre-existing strand to add these nucleotides
o DNA is eteded i a ’ to ’ dietio
- Sequence-specific primers (oligonucleotides) must flank DNA sequence needed for
o Forward and reverse primer
- Nucleotide bases (dNTPs): dATP, dCTP, dTTP, dGTP
- Reaction buffer with essential cofactors (dependent upon DNA polymerase requirements)
o HCl for Taq
o Optimal pH
o MgCl2: 1-2.5 mM
o Salt (e.g. KCl): ionic strength important
o DMSO: Facilitates strand separation for larger pieces of DNA disrupts base pairing
o Test a range of cofactor concentration and range of annealing temperatures
- Thermal cycler
Oligonucleotide Primer
- Oligo, primer, probe
- Short sequence of DNA (approx. 15-25 bases)
COMPLEMENTARY to the target sequence
- Will hybridize (anneal) to single stranded DNA
o Temperature specific: Tm
- Used  DNA polease to sthesize DNA ’
to ’
Primer design considerations
- Increasing primer length can theoretically increase specificity:
o 16 base: 416 = 1 in 4,294,967,296 chances of annealing
o 20 base: 420 = 1 in 1,099,511,627,776 chances of annealing
- Reduce the number of mismatches to increase specificity
o Primer should be complementary to the original strand
- ’ ed of pie should ed i G o C: stegth of ase-paiig at the ’ ed of the pie is critical:
o This end needs to be the most stable
o ’ does’t eed to e so stale, ad hee estitio sites a e itodued
- GC content:
o 40-60%
o Affect the Tm
- Secondary structure, avoid:
o Hairpin (annealing within primer
structure)
o Primer-dimer (two primers annealing
together)
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