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components of PCR
- Thin-walled tube
- For efficient & homogenous heat transfer.
- Template DNA
- Contains the target sequence to amplify.
- Nucleotides (dNTPs)
- Mixtures of nucleotides, which are building blocks of new DNA strands.
- PCR buffer & H2o
- Creates optimal conditions (ionic concentrations & pH) for optimal activity of Taq polymerase
- Magnesium chloride (MgCl2)
- Essenstial cofactor for DNA Polymerase
- Primers (Forward & Reverse)
- Short, single strand DNA, Provide specificity of PCR by selecting the DNA region to be amplified.
- DNA Polymerase
- Enzyme catalyzing the polymerization of dNTPs into DNA strands.
Setting up a PCR rxn
- • Keep reagents on ice to avoid non-specific amplification.
- • Prevent contamination (from pipet barrel or fingers)
- • Negative control: verifies contamina+on did not occur.
- • Positive control: verifies PCR reaction worked.
- • Tm = Annealing temperature + 5°C
- • Tm of the two primers need to be similar.
- • 50°C < Tm < 65°C
- • Tm = [4°C x (G+C)] + [2°C x (A + T)]
- • Primer length should be 17 – 28 bp (ideally 20 bp)
- • Primer GC content should be 40-60%
- • GC clamp (1-2 G or C at the 3’ end of primer) increases
- specific binding.
- • Ideally pure, unbroken DNA
- • Optimal concentration
- • 50 ng – 500 ng for gDNA
- • 50 pg – 50 ng for pDNA
Types of PCR
Realtime (quantitative PCR) Allows quantitation of initial DNA in a sample by using fluorescent probes.
Reverse Transcription PCR Uses reverse transcriptase to reverse mRNA into cDNA before the actual PCR reaction begin.
- Multiplex PCR Detects multiple target sequences simultaneously
- on the same staring DNA material.
Stages of PCR
- 95 C
- 5 min intial + 1 min
- Temperature is increased to separate DNA stands.
- Ta:50-65C Ta= Tm of primer - 5 C
- :30-1 min
- Temperature is decreased to allow primers to base pair complementary to DNA template.
- 1min/1kb of template
- 72 C
- Polymerase extends primer to form new DNA strand
Agarose gel electrophoresis
serparate DNA fragments by size using an electric field
The concentration of
agarose is determined by
the size of the fragments
- Large fragments (>1kb): 0.7-1% (m/v)
- Smaller fragments (<1kb): higher %
- agarose (1.5-2%).
- Most common conductive buffers for DNA electrophoresis:
- – Tris/acetic acid/EDTA (TAE)
- – Tris/boric acid/EDTA (TBE)
“Next generation DNA sequencing”
- • PCR-based
- • Does not require electrophoresis
- or fragment separation step
- • Faster than Sanger method
- • Only generate up to 150 bp
- • Hundreds of thousands reads in a
- single run
Real-time (quantitative PCR)
Allows the quantitation of initial DNA in sample by using fluorescent probes.
Rerverse transcription PCR
Uses reverse transcriptase to reverse mRNA into cDNA before the actual PCR reaction begin.
Detects multiple target sequences simultaneosly on the same starting DNA material.
Amplifies DNA when the information about the target sequence is limited or when the same PCR needs to work with DNA templates from different species.
Uses 2 rounds of PCR with 2 sets of primers to avoid non-specific amplification
Random Amplification of Polymorphic DNA
- Fingerprinting method used when the gDNA
- template sequence is not known.
- calculated from the number of gaps and
- substitutions associated with each aligned sequence.
- The higher the score, the more significant the alignment.
E-value (expected value)
- Likelyhood that a
- sequence with a similar score will occur in the database
- just by chance. The smaller the E-value, the more
- significant the alignment.
Accession number links
- providing direct access from
- BLAST results to related entries in other databases.
- • E-values < 10-4 : significant homology
- • 10-4 < E-value < 10-2 : should be checked (similar
- domains but maybe non-homologous)
- • 10-2<E-value< 1: no good homology
Similar genes with similar function are (share the same ancestor)
molecular differences in homologous genes (or proteins) are directly correlated with evolution time.
current day species
- hypothetical most recent common
- “elapsed time” from one
- speciation to the next
Weighted (scaled) Tree
- the relative lengths of the branches
- represent the amount of genetic
- changes (and also time).
Unweighted (unscaled) Tree
- the branches have arbitrary equal
- occurs when the
- similarity does not derive from
- shared ancestry.
- Tree based on the total minimum
- number of character changes
- between the nodes.
- • The tree is built with the fewest
- substitutions required to explain
- the difference observed in data.
- • “The simplest of two explanations
- is preferable”
- Based on finding the tree that is
- the most most likely to generate
- the sequence data observed.
- • Use an evolutionary model of
- how character states change.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
- Both create a banding pattern
- unique for each individual.
1. DNA containing the gene of interest is extrcted from human cells and cut into fragments by restriction enzymes.
2. The fragments are separated according to size by gel electrophoresis. Each bands consists of many copies of a particular DNA fragment. The bands are invisible but can be made visible by staining.
3. The DNA bands are transferred to a nitrocellulose filter by blotting. The solution passed through the gel and filtered to the paper towels.
4. This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel.
5. The filter is exposed to a radioactively labeled probe for a specific gene. The prode will base-pair (hybridize) with a short sequence present on the gene.
6. Th filter is then exposed to x-ray film. The fragment containing the gene of interest is identified by a band on the developed film.
PCR based methods
- STR loci are targeted with sequence-specific primers and are amplified using PCR
- • Multiplex PCR allows to amplifies several targets simultaneously
- • DNA profiles are visualized by gel electrophoresis
RFLP & PCR
PCR is shorter, required less DNA, DNA could be degraded, descrete alleles obtained, DNA can be either single stranded or DS, autotable and capable of high-volume sample processing
- “Techniques of genetic recombination used to bring
- together genetic material from multiple sources,
- creating sequence that would NOT otherwise be found
- in biological organisms”.
origin of replication
- is the starting point
- for replication.
- To express a gene, a
- plasmid must have a
- promoter upstream of the
- coding region.
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