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EB 3410 Chapter 6
Manipulating Biomolecules: DNA; Polymerase Chain Reaction (PCR)
| Question | Answer |
|---|---|
| Why is there variability in the intronic regions of genomic DNA? | SINEs; endonucleases insert transposable elements; SNPs |
| When did Kary Mullis develop PCR? When did he earn the Nobel prize? | developed: 1983 and earned Nobel prize: 1993 |
| What are the 4 areas of biotechnology PCR impacted? | Gene mapping, cloning, DNA sequencing, and gene detection |
| How has PCR been used in medical diagnostics? | Detect disease causing mutations, identify criminal suspects, and sequence the human genome |
| T or F: Prior to PCR, it would have been impossible to do forensic or genetic studies with this small amount of DNA | True |
| PCR produces _____ large amounts of a specific piece of DNA from trace amounts of starting material (________) | exponentially; template |
| What materials are needed for DNA extraction? | Saline mouthwash, InstaGene matrix, 56oC Water Bath, 100oC water bath |
| What is the purpose of the saline mouthwash? | 1.Collecting cells 2.Isotonic Solution-to keep DNA intact |
| How large should the cell pellet be after centrifugation of the saline wash? | about the size of a match head |
| What is the purpose of Chelex microscopic beads? | Binds divalent cations; prevent/inhibit DNases/nucleases |
| Why is it important to bind divalent cations? | prevent DNases from working (degrading DNA) |
| What is the purpose of the 56oC water bath? | (1)Breaks connective tissue holding cells together (2)Inactivates (denatures) DNases |
| What is the purpose of the 100oC water bath? | Rupture Cell Membranes (1)DNases will be inactive (2)Free DNA in tube |
| Why does the InstaGene matrix need to be removed prior to PCR? | The divalent anions/microscopic beads may inhibit the PCR reaction |
| How many pairs of chromosomes do humans have? | 23 pairs |
| ___% is coding DNA and ___% is noncoding DNA | 5% active (introns and exons) and 95% inactive |
| How many genes do humans have? | 30,000 - 50,000 |
| What is needed for a PCR rxn? | Template DNA; Master Mix (Primers, dNTPs, Taq polymerase, reaction buffer (with MgCl2)) |
| Where does Taq polymerase bind? | Free 3'OH group |
| What are SINEs? | Intronic Sequences that have been the target of random insertions |
| About how many bp is the Alu sequence? | 300bp |
| How many times is the Alu sequence repeated (one copy at a time) | 500,000 times |
| What is the origin/function of the Alu sequence? | UNKNOWN |
| What is the Alu sequence named after? | A microbe with a similar sequence: Acobacter luteus |
| Why is the Alu element important? | Useful to genetics: if present within introns of genes associated with pathogens/disease; estimate relatedness |
| T or F: Analysis of Alu repeats is a simple measure of molecular genetic variation - with no reference to disease or relatedness among individuals | True |
| How is the Alu repeat dimorphic | Can be present or absent on each chromosome (+/+, +/-, or -/-) |
| What are the stages to set on the thermal cycler? | Initial denaturation; denaturation; annealing; extension; final extension; hold |
| What is the temperature and duration setting for the -Initial denaturation stage? | 94 degrees C; 5min |
| What is the temperature and duration setting for the -denaturation stage? | 94 degrees C; 1 min |
| What is the temperature and duration setting for the - annealing stage? | 52 degrees C (based on primers); 1min |
| What is the temperature and duration setting for the -extension stage? | 72 degrees C (ideal for Taq); 2min |
| What is the temperature and duration setting for the -final extension stage? | 72 degrees C; 6min |
| What is the temperature and duration setting for the - holding stage? | 4-15 degrees C; infinity! |
| Why is the loading dye added to the sample (when loading in a DNA gel)? | add weight to sample; track migration in gel |
| what are the benefits of using DNA fast blast stain or UView? | Nontoxic and noncarcinogenic; waste generated is not considered hazardous; relatively inexpensive |
| Explain amplification of DNA in PCR with an equation. | copies = X(2^n) ---x=copies of template DNA and n=number of cycles |
| On an electrophoresis gel, a band _______ bp represents +Alu and a band _______bp represents -Alu | 960bp +Alu; 660bp -Alu |
| What is the Hardy-Weinberg equation? | p^2 +2pq +q^2 = 1 |
| in the hardy-weinberg equation; p = _______ and q = _______ | p=frequency of + allele and q=frecency of -allele |
| In the hardy-weinberg equation p^2=_____; 2pq=______; and q^2=_______ | frequencies of +/+, +/-, and -/- respectively |
| What is the equation for calculating the genotypic frequency? | Number of genotypes/Population Total (N) |
| What are some techniques based on PCR? | DNA microarrays; DNA sequencing; Medicine; Agriculture; Forensics; Paternity test; human migration; wildlife conservation |
Created by:
savelae
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