Terms in this set (78)

• PCR Uses a DNA Polymerase to Amplify Selected DNA Sequences in a Test Tube
• Multiple Cycles of Amplification In Vitro Generate Billions of Copies of the Desired Nucleotide Sequence
- Used to Obtain either Genomic or cDNA Clones
1. Isolate total DNA/mRNA from cells
2. DNA/mRNA sequence to be cloned
3.
DNA:
1. Separate strands (by heating) and add primers (cool to anneal)
2. PCR Amplification
3. Genomic Clones

mRNA:
1. Add First Primer, Reverse Transcriptase, and Deoxyribonucleoside Triphosphates
2. Separate strands (heat) and add second primer (cool)
3. PCR Amplification with Both Primers Present
4. cDNA Clones

• PCR is Also Used for Diagnostic and Forensic Applications
A. ex: determine if infected with HIV
1. blood sample from person
- Remove CElls by Centrifugation
2. Rare HIV particle in plasma of infected person
- Extract RNA
3. Reverse transcription and PCR Amplification of HIV cDNA
4. Gel Electrophoresis with infected sample and control (using blood from noninfected person)

B. Forensic Science - distinguish individuals
- DNA sequences analyzed are short tandem repeats (STRs) composed of repeating 2 letter sequences - number of repeats in population is highly variable - hereditary
A) PCR using primers that recognize unique sequences on either side of one particular STR locus produces a pair of bands of amplified DNA from each individual, one band representing maternal and other paternal STR variant
- different people can have several bands in common, but overall pattern differs - band can serve as DNA fingerprint!
- more loci = more confidence - decreasing likelihood of sharing same fingerprint by chance -- also used in paternity test
A surface on which a large number of short DNA fragments (typically in the tens of thousands) immobilized in an orderly pattern. Each DNA fragment acts as probe for mRNA produced by a specific gene, allowing the exp of every gene in a genome to be monitored
1. mRNA collected from different samples - can be used to look at diff genes, species, tissue types, or cancer cell vs normal cell if you are searching for cancer disease genes up-regulated/down regulated, etc.
2. each is reverse transcribed to cDNA complementary to mRNA, with different color labeled fluorochrome
3. cDNA hybridizes to DNA samples in microarray (incubate)
4. Wash, scan for fluorescent color signals and combine images
- cDNAs complementary to type A mRNAs hybridize with DNA (in array) complementary to type A cDNA
- if cells don't exp any mRNAs that correspond to cDNAs, no cDNAs are made that binds to those mRNAs
- typical cell exp's 100s-1000s of mRNAs - 100s-1000s of spots ID'd - study many genes simultaneously from a particular type of cell, or cells exposed to environmental condition
- Automated fluorescence microscope determines which mRNAs were present in the original sample based on the array positions to which the cDNAs are bound
- 1 drawback: sequences of mRNA samples to be analyzed must be known in advance and represented by a corresponding probe on the array
- color shown = gene in sample that is expressed at a higher level than the corresponding gene in the other sample
- mix of 2 colors - equal level of expression of genes in both cell samples
- intensity = how much RNA present from a gene
- dark spot = little/no expression of gene whose fragment is located at that position in the array
- represents lots of genes - allows for direct comparison of specific genes expressed under both conditions
- 1 dot = 100,000 bp of human genome
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