Download Chapter 8. Manipulating DNA, RNA and proteins

Chapter 8: Manipulating DNA,
RNA and proteins
Isolating the correct tissue
Analysing DNA in tissues
Accuracy with numbers
• Blond hair
Accuracy with numbers
• Blond hair
• Female
Accuracy with numbers
• Blond hair
• Female
• Blue pants
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Pony tail
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Pony tail
White shirt
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Pony tail
White shirt
Short sleeves
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Pony tail
White shirt
Short
sleeves
• Glasses
Accuracy with numbers
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Blond hair
Female
Blue pants
Black shoes
Pony tail
White shirt
Short
sleeves
• Glasses
• Under 5’5”
Measuring and changing gene expression
How much RNA is there?
RNA quantification
How can the levels of RNA be altered?
Increasing and decreasing RNA levels
Assessing rates of transcription
Reporter genes
Quantifying RNA
Methods are available to measure RNA levels:
-within the cell (in situ hybridization)
In situ hybridization of
fluorescent cDNA to
bind to mRNA
(presence or absence
of expression)
Quantifying RNA
Methods are available to measure RNA levels:
-within the cell (in situ hybridization)
-in tissue samples (northerns, DNA arrays)
Tissues must be broken and the integrity of RNA
preserved by inhibiting RNA degradation by
RNases
RNA is purified (several ways) and quantified by:
-northern hybridization
-reverse transcription -PCR (RT-PCR)
-cDNA arrays
Quantifying RNA
Northern blots: electrophoresis to separate
RNA, transfer to membranes, hybridize with a
single radioactive cDNA
Quantifying RNA
RT-PCR: ultrasensitive way to
measure levels of
specific RNAs
1 2 5 10 20 50pg
Product
2ul
Real-time PCR: very rapid
(and sensitive) way to
measure mRNA by
combining fluorescent
detection with PCR
10 pg RNA in the
2ul unknown
# of PCR cycles
Quantifying RNA
cDNA arrays:
compare 1000’s of
RNA’s in treatment
pairs (ie control vs
treated)
Bioinformaticists help interpret vast
amounts of array data
1 2
-genes are grouped
into characteristic
patterns
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Proteins
Altering gene expression
(1) Prevent protein from working
-add inhibitors
-microinject antibodies
-dominant negative transgenes
Altering gene expression
(2) Reducing RNA levels by enhancing RNA
degradation or blocking translation
-anti-sense (injections of RNA, oligonucleotides
or morpholinos)
-RNA interference (RNAi)
Altering gene expression
(3) Alter genes
-random mutagenesis
-targeted knock-outs
-gene replacement or addition
Transfecting cells, including bacteria
Living bacteria (and other cells) can be transfected
with DNA
Cells can be treated to enable them to take up DNA
(competent cells)
Cells can be permeabilized by high voltage
(electroporated) to take up DNA
DNA can be packaged in liposomes that get
incorporated into membranes
DNA can be packaged into viruses that infect cells