How DNA sequence data is obtained for genomic research?
- Obtain Samples
- Purify the DNA
- Copy your Gene
- Make sure your gene is copied
- Obtain sequence data
DNA Purification Using “Spin
Columns”
- Chop up tissues and break open the cells with detergents
- Separate the DNA from the rest of the cell debris using spin column and centrifugation
- Suspend the DNA in buffer for future use
Common challenges in DNA extraction
- Sample Type
- Sample Preparation and Storage
- Choosing the Right Method
Polymerase Chain Reaction (PCR)
technique allowing the exponential amplification of small quantities/specific regions of DNA utilizing thermal cycling and enzymatic reactions
PCR is developed by
Kary Mullis in 1983
PCR components
- Template (DNA)
- Primers
- DNA polymerase (Taq)
- dNTPs (deoxynucleotide triphosphate)
- Buffers
PCR Process
- Initialization
- Denaturation
- Annealing
- Extension
- Repeat
- Final Elongation
- Hold
Choosing primers:
- Should be 18-25 nucleotides in length
- Calculated melting temperature varies depending on the method used (55-65°C), but should be nearly identical for both primers
- Avoid inverted repeat sequences and self-complementary sequences in the primers, avoid complementarity between primers (‘primer dimers’)
- Have a G or C at the 3’ end (a G/C “clamp”)
Sources of problem in PCR:
- Inhibitors of the reaction from the template DNA preparation (protease, phenol, EDTA, etc)
- Cross-contamination by DNA from sources other than the template added
if this becomes a problem:
- Work in a laminar flow hood (decontaminate using UV light 254 nm)
- Use PCR dedicated pipettors (with barrier tips), PCR dedicated reagents
- Centrifuge tubes before opening them to prevent spattering, pipet contamination
Types of PCR:
- End-point PCR
- Reverse-Transcriptase PCR (RT-PDR)
- Multiplex PCR
- Quantitative of real-time PCR (qPCR)
End-point PCR, end products?
are visualized on an agarose gel to determine their size as well as relative quantity
End-point PCR, is used for?
applications such as cloning, sequencing, genotyping and sequence detection
Endpoint PCR is ____ ___ ____ than real-time PCR
far less quantitative
End-point PCR is mostly used to
- detect presence or absence of targets
- but can also be used to estimate relative quantity
Reverse-Transcriptase PCR (RT-PCR)
technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by reverse transcriptases
Multiplex PCR
- used for the amplification of multiple targets in a single PCR experiment
- it amplifies many different DNA sequences simultaneously
Quantitative or real-time PCR (qPCR)
- the DNA amplification is detected in real-time with the help of a fluorescent reporter
- the signal strength of the fluorescent reporter is directly proportional to the number of amplified DNA molecules
Agarose gel electrophoresis
- Determine the required gel percentage
- Pour the gel
- Mix samples/ladders with loading dye
- Load the gel
- Run the gel
- Visualization
Can you identify the purpose of the polymerase chain reaction?
It is a DNA amplification technique
DNA extraction from plant tissues is difficult due to:
Presence of secondary metabolites and polysaccharides
DNA extraction requires _________to lyse the epithelial cells
and to degrade compounds inhibitory to amplification
Heat treatment
In the agarose gel the DNA migrates in the following direction:
negative to positive pole
The ratio of absorption at 260nm to absorption at 280nm is commonly used to assess:
The purity of DNA and RNA with respect to protein
