Exam 2 (PPT 1) Flashcards
PCR Primer Design
Process (2) and Principles (6)
1) Find just before and after desired gene sequence
2) Anneal primers to isolate gene during extension
Principles…
1) Primer Length: usually 18-22 bases
2) Primer Melting Temperature: 52-58°C
3) GC Content: Should be 40-60%
4) Start and end with 1-2 G/C bases
5) Avoid some secondary structures such as Hairpins, Self dimer, and Cross dimer.
6) Primer pairs should not have complementary regions
The Procedure of PCR Amplification
1) Denature DNA
2) Anneal Primers
3) Extend Primers
*usually 18-35 cycles
How many copies does PCR make in 18 cycles? in 35?
2^18 =262,144
2^35 =34,359,738,368
Electrophores
“Gel electrophoresis”
*Can be agarose or acrylamide
Gel electrophoresis =
Electrolytic cell
How is genomic DNA resolved?
Pulse-field gel electrophoresis
because its too large to be resolved by normal gel electrophoresis —> The MW limits for normal Gel electrophoresis: 30-50KDa
Cloning
restriction endonucleases and DNA ligation
sticky end vs blunt end (sticky end allows the DNA fragment to be inserted into a plasmid vector)
What are 3 enzyme reaction conditions?
B.E.T
1) Buffer
2) Efficiency
3) Temperature
Enzymes perform differently when these conditions are altered. Companies tend to recommend the optimal conditions for max enzyme functionality.
What does the term “units” refer to when ordering enzymes?
“The amount that catalyzes the conversion of 1 micro-mole of substrate per minute. “ -Prof. Ye
which means (…?)
Southern Blot hybridization
“Southern blotting”
“method used for detecting a specific DNA sequence”
“combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.”
-Prof Ye
Northern Blot hybridization
plus steps
“Northern blotting”
“used to detect RNA (or isolated mRNA) in a sample”
Steps:
1) RNA is extracted from a sample
2) Electrophoresis seperates the RNA by size
3) Northern blotting transfers RNA to membrane
4) Desired sequences of RNA is targeted by labeled probes
5) Labeled RNA is visualized on X-ray film
Eastern Blot hybridization
“a biochemical technique used to analyze protein post translational modifications (PTM) “ Prof Ye
i.e: lipids, phosphates, glycoconjugates, but mostly CARBOHYDRATE EPITOPES***
How is DNA fragments cloned into a Plasmid Vector?
The fragment is ligated into a plasmid vector that has a drug resistant gene. This RECOMBINANT plasmid vector is then transformed into the Ecoli cell where only the successfully recombined plasmid vectors will grow colonies on a drug plate that the plasmid confers resistance to.
mRNA has what kind of tail?
Poly-A tail
*what is happening in slide 22? Is this how the sticky end is created?
When it comes to chain terminating sequences, ____ is changed to ____.
What is the difference b/w them?
dNTP changed to ddNTP
deoxy changed to dideoxy
OH on 3’ changed to H on 3’
*the oxygen is what allows for the addition of the next nucleotide because it attacks the 5’ position of the next nucleotide. Without the oxygen, the chain is terminated.
Most Popular Sequencing Method
Fluorescent Chain-terminating Nucleotides
A type of Chain Termination method
Normal Sanger sequencing can only sequence 100 to 1000 bp. What should we do if we want to sequence a longer DNA?
SHOTGUN sequencing (SLOW!)
“Linking Contigs by Sequencing the End of Large DNA Fragment”
“Paired-End Strategy Permits the Assembly of Large-Genome Scaffolds”
- Prof Ye
Next Generation Sequencing
sequences 200-500bp
But way FASTER sequencing
Bioinformatics Tools are important for Annotation. What is Annotation?
Annotation: is the systematic identification of every stretch of genomic DNA that contains protein-coding information or non-coding sequences that specify regulatory RNAs such as microRNAs.
Ion exchange chromatography vs Gel filtration chromography vs Affinity chromatography
Ion Exchange: charge separation
Gel filtration: Size separation
Protein Affinity: immunoaffinity (HIS tag + ect)
polyacrylamide gel electrophoresis (PAGE).
Seperation based on size if SDS + BME used
Western Blotting
Immunoblotting
“Useful for determining expression levels of specific proteins.”
“Makes use of antibody biology”
Edman Degradation
Protein sequencing that uses PITC to label and degrade the peptide, reducing the chain by 1 residue each time.
Tandem Mass Spectrometry (MS/MS)
1) Protein extraction from cell
2) digest with sequence specific protease
3) Separation via ion exchange chromotagraphy
4) Reverse phase column (non polar = stationary phase)
5) Peptide sequences analyzed
