DG Exam 1 Review PDF

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DG Exam 1 Review ● Gene structure = introns (non-coding sequence) & exons (coding sequence that is expressed for protein synthesis) I. Pipette/Units P2

P20

P200

P1000

.2-2µl

2-20µl

20-200µl

200-1000µl

● 1µl=10^-6L ● 1mL=10^-3 ● 1µl=10^-3mL II. PCR - isolates & amplifies specific regions of DNA ● Requires DNA polymerase, nucleotides, template, & primers to replicate DNA 1. Denaturing (hydrogen bonds separate strands) 2. Annealing (primers bind to complementary nucleotides on single-stranded DNA template) 3. Extension (Taq extends the primers, synthesizing new strands of DNA) Genetic Information Transfer (gene expression) “Central Dogma”

● Genomic DNA= introns & exons (longer sequence) ● cDNA= exons (shorter sequence) 1) Understand the function of PCR primers… ● PCR primers are made of DNA ● 20 nucleotide sequences ● Single stranded molecules ● Forward and reverse primers designed to target template sequence that you want to amplify ● During annealing, primers bind ● Nothing beyond the primers range in copied

2) Design a PCR reaction table… ● V(T) = total volume per reaction EX: 6 DNA samples 1 Reaction

8 reactions

Water

w/o DNA-2(primers)-5(Taq)=9

72

4X Taq use C1V1=C2V2

4X?=1X(20)=5

40

Primer F

0.5

4

Primer R

0.5

4

Master Mix w/o DNA

(Total-DNA)= 15

120

DNA

5

---

Total

20

---

III. ●

● ● ● IV. ● ● ● ● V.

VI. ●

To find # of reactions= n(DNA samples)+2(negative control & slush) Gel electrophoresis DNA is negatively charged ○ Sending currents through it will make it travel to the positive side ○ Red (+) ○ Black (-) Water is always the negative control ○ Shows band = contamination/something wrong w/ Master Mix Positive control allows the experiment to be conducted giving off results to be interpreted Ladder= estimate size of fragments; migrates in same order Use BLAST to search a DNA database… BLAST helps find the similarities between sequences Find where introns & exons are located to see what gene looks like MUSCLE is used to search for multiple sequences Help find location of polymorphisms Polymorphisms A. SNPs (single nucleotide polymorphism) = difference in 1 nucleotide B. Indels (insertions/deletions) = 1 or more nucleotides of DNa is present in 1 individual & absent in another C. TEs (transposable elements) = moves to another position in genome; long sequence is missing from one strand of DNA Bacterial Transformation GFP (green fluorescent protein)= gene that allows bacteria to glow green under blue UV light ○ Useful for tagging proteins of interests

● Bacteria is transformed w/ plasmid that contains GFP ○ Plasmids are introduced to bacteria, bacteria amplifies plasmids creating larger quantities ● GFP is clonded from jellyfish ● Plasmid= genetic structure in a cell that can replicate independently of the chromosomes ○ Plasmids are mixed with bacterial cells treated w/ calcium chloride to ensure it’s capable to take up DNA ● Gene promoters= initiates the expression of the gene ○ Constitutive promoter= always on ■ EX: amplicion resesitance gene ○ Conditional promoter= not always on, needs a ■ EX: GFP promoter needed bind w/ arabinose to express the gene ● Ampicillin fights bacteria cells; no DNA = no formation of colonies ● Expression of gene= ● Presence of gene= ● Transformation efficiency = (# colonies/5µg of plasmid)(500µl transformants/100µl plated) VII. Vocabulary ● DNA = carrier of genetic information ● mRNA= attaches to ribosomes & identities protein structure ● pre-mRNA= unzippint DNA makes pre-mRNA ● tRNA= attached to amino acid that brings in anticodons to codons on mRNA ● rRNA= makes polypeptides during translation in ribosome ● Spliceosome (splicing)= cut out of introns to leave only exons ● cDNA= copy of RNA made through reverse transcriptase ● Ribosome= location for protein synthesis ● Reverse transcription= RNA -> DNA ● Reverse transcriptase= enzyme that makes cDNA copy of mRNA. ● Protein= composed of amino acid polymers joined together by peptide bond ● DNA polymerase= enzyme that synthesizes new copies of DNA ● RNA polymerase= enzyme that makes RNA from DNA template...