molecular diagnostics exam 1

in one nucleotide of DNA what are the 3 major moieties (components) that make it up?

- 5 carbon sugar - phosphate group - nitrogen bases

5. What two methods can you use to quantify extracted DNA?

-Use a spectrophotometer - 260 and 280 nm. -Run DNA in an agarose gel. Note: to quantify your band you need a special ladder with not only known sizes, but also know concentration of DNA in each band (hence different intensities); these are commercially available but more expensive.

state 2 additions to the standard PCR machine that make it a real-time PCR machine. these differences explain why it is more costly

-need additional computer software and database - need additional optics to detect fluorescentce

what are the three chemical difference between DNA and RNA?

-sugar -base -single/double strand

List the reagents needed to set-up a real-time PCR reaction

-taq DNA polymerase -buffer -template DNA -2 primers -dNTPs -fluorescent probe

1. How do DNA and RNA differ chemically and structurally?

1) On sugar: RNA has an OH on C-2' (ribose), and DNA has an H on C-2' (deoxyribose). 2) Nitrogenous base: RNA uses uracil in the place of thymine. 3) RNA is typically single stranded, DNA is double stranded.

a PCR machine cycles between 3 temperatures "steps". what is the name of each step and what is occurring during that step 1- 94 degrees c 2- 55 degrees c 3- 72 degrees c

1- denaturation, melting of DNA to become single stranded 2- annealing, hybridization of primers 3- extension, polymerization of DNA

1. What reagents are mixed together in a tube to run a PCR reaction?

1. Taq DNA Polymerase 2. 2 primers 3. dNTPs 4. template DNA 5. buffer

list two differences between mtDNA and chromosome DNA?

1. circular mtDNA 2. in mitochondria 3. passed down by mother 4. mtDNA is smaller

Name 2 applications of restriction enzymes

1. clonning 2. DNA fingerprinting (RLFP)

list three things needed for transcription: RNA synthesis gene expression

1. enzyme: RNA polymerase 2. DNA template: antisense strand 3. RNA synthesis is 5' to 3'

what reagents are added to the tube to set-up a restriction digestion reaction

1. extracted DNA (in agarose plugs) 2. DNA water 3. buffer 4. restriction enzyme

name 3 differences between DNA and RNA

1. on sugar, RNA has an OC on C-2' (ribose), and DNA has an H on C-2' (deoxyribose) 2. nitrogenous base: RNA uses Uracil in plase of thymine 3. RNA is typically single stranded and DNA is double

list enzymes used in DNA in vivo replication but not in PCR reaction in vitro?

1. topoisomerase 2. helicase 3. primase 4. ligase

if you wanted to cut a realitivly short DNA fragment into as many bands as possible, would you choose 4, 6, or 8 bp cutter to test

4 bp cutter (not sure what this is or if its important lol)

The following questions relate to the restriction enzyme HpaII: HpaII cut site: 5'....C^CGG....3' 3'....GGC^C....5' is HpaII a 4, 6, or 8 bp cutter enzyme

4 bp cutter enzyme

5. Which direction is a DNA sequence always written in a) 5'-3' or b) 3'-5'? Why

5'-3' direction. If it was not standardized this would lead to incorrect interpretation of protein sequences and presentation of sequence data. Replication always occurs in the same direction in the cell.

why does step 3 always occur at 72 degrees c?

72 degrees is the prime temperature that taq DNA polymerase can polymerize DNA, the extension temp never varies

1. What changes are made to standard PCR to make it Real-time PCR? Note: this is the method most frequently adopted for clinical diagnosis. How is the machine different? What changes are made to the reaction mix?

A Real-time PCR machine is a thermocycler, plus it has optics to detect fluorescence (of the probe). It also has the needed software on an attached computer to report the results. It is a more expensive an elaborate machine then a standard thermocycler. The reaction mix is the same as traditional PCR except you need to add some a way to get the DNA to fluoresce - either by adding SyBr Green dye or a fluorescently labeled probe (there are multiple options for the probe - e.g. FRET, TaqMan, Molecular Beacon).

8. What is a plasmid, and how does it differ from chromosomal DNA?

A plasmid is an extrachromosomal (not extraneous), circular piece of DNA found in bacteria. Plasmids are easily transferred between cells and often carry antibiotic or heavy metal resistance genes to help bacteria better adapt to a wide range of environments. They have been used extensively in molecular biology as a tool to move genes between organisms (cloning).

3. Based on hydrogen bond pairing, which would require a higher temperature to melt, a DNA region rich in A-T or G-C?

A rich GC region has a higher melting temperature (due to the 3 hydrogen bonds in GC pairs verses the 2 hydrogen bonds in AT pairs).

4. Some companies have developed non-PCR based methods to detect specific pathogens. Hybrid Capture Assay is an example. What is the basic principle of this method? List advantage(s) of this over PCR?

Advantage: Does not require a PCR machine (isothermal method) so cheaper Hybrid Capture is based on hybridization rather than target amplication. You mix your sample DNA (make it single stranded before starting) with a probe to your gene of interest. If the probe attaches to the sample it will become double stranded, and the rest of the sample will stay single stranded. Then the mixture is added into a well of a 96-well plate that is coated with antibodies that bind double stranded DNA, trapping your DNA of interest onto the sides. Wash to remove the single stranded DNA from the sample. Then add another antibody specific for dsDNA, but this time have it labeled for detection (sandwich assay). Typically detection is with a light emitting reaction, and the sample can be read on a luminometer.

name a method that uses the fact that DNA is charged to get that method to work?

Agarose gel electroporesis

dNTPs

DNA building block

what is the overall charge of DNA and on which moiety is it found

DNA is overall negative, it is found on the oxygen of phosphate backbone

8. What is the purpose(s) of including a DNA ladder in an agarose gel?

DNA ladders contain bands of known sizes that can be used as a reference to compare with the sizes of your DNA fragments. It also can be used a positive control to make sure your gel worked

9. What dye is added to visualize DNA in an agarose gel?

Ethidium bromide or CyBr Green. The later is less hazardous but more expensive.

4. Would you expect a hypothermophile (bacterium or Archaea that lives in very hot temperatures) to have a low or high GC content? Why?

Expect a higher GC content to prevent their DNA from becoming single stranded at the high temperatures they live in.

list 3 genome-wide sequencing projects in identification and annotation human disease after the completion of human genome project:

Hapamp GWAS 1000 genome (TCGA) human microbiome project cancer moon shot project

draw what the electrical field looks like in PFGE and diagram how it changes in time

It is a hexagon shape with + on one side and - on the other. the electrical field moves from - to + direction but doesnt have one swift motion, it bounces around between the different sides

3. One final step of a DNA extraction is to add alcohol. Why

It precipitates the DNA out of solution. That way the DNA can be concentrated, by then re-dissolving it in a much smaller volume of water or buffer.

2. The Phenol chloroform method is a common liquid-phase extraction method. When you add the phenol:chloroform you get two layers (aqueous and organic) and the DNA is in the upper layer (aqueous). What is the purpose of this step?

It separates the DNA from other cellular debris like proteins, lipids and carbohydrates

6. What is the difference between the leading strand and a lagging strand in DNA replication?

Leading strand - the continuous strand copying along the 5'-3' direction of the opening double helix. Lagging strand - discontinuous copying of DNA in 5'-3' direction on the stand moving in the opposite direction of the opening double helix. The fragments generated in this process are called Okazaki fragments and require DNA ligase to connect them together

2. What is the overall charge of DNA (negative or positive)? Where on the DNA structure does the charge come from?

Negative charge- this is due to the oxygen on the phosphate backbone.

if you were instead extracting DNA from human blood cells, would you need to add lysozyme? briefly state why or why not?

No because the human blood cells do not contain a peptidoglycan cell wall

11. What is the resolution of an agarose gel (ballpark size of fragment in base pairs)? What is the resolution of polyacrylamide?

One textbook I read said 20 bp to 10,000 kbs. My experience is that 500 bp is the lower resolution for agarose (not 20). In general you need about a 100-300 bp difference between bands to see them as two separate bands (this is partially determined by your agarose gel concentration). You definitely cannot resolve single or small bp differences between bands because they would appear as 1 band. Also, if you wanted to compare extremely large fragments above 20,000 bp you can still use agarose, but need a different machine to run the gel. This method is called Pulse Field Gel Electrophoresis and we will cover it in class. If you use polyacrylamide gels you can get single base pair resolution of bands.

one difference?

PFGE detects very large dna band sizes while standard detects normal to smaller sizes

3. There are several different methods used to detect product (amplicons) in a real-time PCR reactions. The simplest is SyBr Green and the rest all use probes (e.g. Taqman, Molecular Beacons, Fret probes). Although it is the easiest and cheapest detection method, list 1 problem with using SyBr Green.

Problems: It is not specific to the amplicon, so all DNA will fluoresce even if it is the wrong product. The probes all hybridize to the amplicon to fluoresce, so they are specific.

NTPs

RNA building block

4. Why is RNA harder to extract that DNA

RNA is less stable because it is not protected by the double helix structure. The ribose sugar is also more easily subject to degradation. Lastly, RNases are more ubiquitous then DNase (they can be found on your fingertips).

1. Why are solid-phase extraction methods (column chromatography) superior to liquid-phase extraction methods (e.g. phenol-chloroform)?

Solid phase extraction methods are 1) relatively easy to use, 2) allow processing of large batches of samples, 3) highly reproducible, and 4) adaptable to automation. They also don't produce organic waste that is difficult to dispose of (but they do produce plastic waste)

2. Define CT value in Real-time PCR? Why is the CT value important (what is it used to calculate)?

The threshold cycle or Ct is the cycle number when the sample fluorescence level crosses the base-line (threshold). It is used to compare with a standard curve and determine the starting concentration of DNA.

7. List the major enzymes needed in DNA replication.

Topoisomerase and helicase- open double helix DNA polymerase - copy DNA strand DNA ligase - connect Okazaki fragements

10. What is the purpose of tracking dye (same as loading dye) and how is this different in function than your answer above?

Tracking dye is used to help ensure that you have successfully loaded your DNA into the agarose gel (it makes the sample visible and contains glycerol so the sample sinks to the bottom of the well). The dye also runs into the gel when the voltage is on, allowing you to track approximately how far your DNA has migrated into the gel. It is different than ethidium bromide in that it does NOT bind to the DNA, so it cannot be used to visualize specific DNA bands.

5. List 3 factors you should consider when designing your own primer pairs.

a. Avoid primers that want to anneal to themselves or other primers b. Choose primers specific to your target, avoiding regions that include repeat sequences. c. Use primers 18-25 bp in length d. Tm of the primers must be the same

7. The use of controls in PCR is very important. How do you set up the negative control (contamination control)? How do you set up the positive control (inhibition control)?

a. Negative control: set up a tube with everything EXCEPT template DNA. In the place of the template put sterile water to make up the volume. If you get a product you know you have contaminating DNA in one of the reagents. b. Positive control: One control is to add a tube with DNA that you know will amplify with your primers. This control ensures your PCR worked fine. Another control is to spike your sample with DNA, usually different from the target sequence you are looking for in your sample. Failure to amplify this control suggests there are some PCR inhibitors in your sample

4. What is the purpose of reverse transcriptase PCR? What is the one addition made to the reaction mix to set up this type of PCR?

a. Purpose: Take mRNA and copy it to DNA (cDNA) so it can then be amplified. b. Addition: Add additional enzyme to PCR tube: reverse transcriptase. Also add Oligo dT primers (for eukaryotic message) or random hexamers or decamers primers. An extra step is added to the PCR reaction to let the reverse transcriptase have time to work (usually at 45-50oC for 30-60 min.).

3. How many cycles is a typical PCR reaction? How long does it take to complete?

a. Typically 30 cycles; takes 30 min-2 hrs depending on machine's ability to change temperatures and the time of the extension step.

6. What steps should be taken in the laboratory to prevent contamination (e.g. errant DNA getting into the sample before amplification).

a. Use a physically separate area for preamplification and post amplification steps b. Use filters in pipette tips c. Prealiquot PCR reagents d. Always run controls

whats the secondary structure of tRNA? a. cruciform or inverted L b. Hairpin c. Triplex d. ring or inverted c

a. cruciform or inverted L

what perspective is consistent with the cancer moon shot project? a. knowing the driving mutations is more important than knowing what organ it arose from, bc different cancers shared the same pathways b. this project the expertise of scientists alone will focus on emerging frontiers in the understanding and treatment of cancers c. the cancer moon shot project will focus on cancer prevention, early detection, not including tumor microenvironment

a. knowing the driving mutations is more important than knowing what organ it arose from, bc different cancers shared the same pathways

choose the best answer: adenine is ____________ a. purine b. pyrimidine c. nucleotide d. dna

a.. purine

what method is always performed following a restriction digestion to check results

agarose gel electrophoresis

under what conditions might you choose to run an agarose gel instead (hint: what can you learn from a gel that you dont get with a spec)?

agarose gel gives you more information about the actual makeup of DNA, you can see the band sizes and know the size of components based on how far they traveled. (check to see if its smeared when you want to see your DNA [?])

what chemical is added to precipitate DNA at the end of DNA extraction protocol so it can be concentrated?

alcohol (ethanol) is added to precipitate DNA to prepare it to be concentrated.

a circular DNA has two BamH1 recognition sites after digestion by BamH1 how many fragments were generated a. 1 b. 2 c. 3 d. 4

b. 2

which of the following is involved in guiding the mRNA export from nucleus to cytoplasm? a. 5' cap b. 3' poly a tail c. sigma d. haRNA

b. 3' poly a tail

Transcription

building block: NTPs enzyme/cellular machine to add that building block: RNA polymerase

Translation

building blocks: amino acids enzyme/cellular machine to add that building block: ribosome

Replication

building blocks: dNTPs enzyme/cellular machine to add that building block: DNA polymerase

what material was sequenced to build phylogenic tree in bacteria a. 18s rRNA b. 23s rRNA c. 16s rRNA d. tRNA

c. 16s rRNA (humans is 18s rRNA)

what concentrations of agarose should be used for optimal seperation of DNA fragments of 100, 250, and 350 bp in length? a. 0.5% b. 0.8% c. 2.0% d. 12%

c. 2.0% small bands= large concentration

a linear DNA has two BamH1 recognition sites after digestion by BamH1 how many fragments were generated a. 1 b. 2 c. 3 d. 4

c. 3

multiple proteins are generated from the same gene by what mechanism a. rna silencing b. operon c. alternative splicing d. histone modifacation

c. alternative splicing

to monitor the progress of electrophoresis, which of the following is added to a sample that doesn not associate with the DNA and runs ahead of the smallest fragments in the sample a. ethidium bromide b. polyacrylamide c. bromophenol blue d. SYBR Green

c. bromophenol blue tracking dye that doesnt bind with dna a- florecent tag typically used for agarose electroporesis b- polymer formed from acrylamide subunits c- asymmetrical cyanine dye used as a nucleic acid stain, binds to DNA (used in PCR)

newly translated peptide was folded by ________ a. helicase b. ribonuclease c. chaperone d. ubiquitin

c. chaperone

genomic imprinting in CpG island is accomplished primarily through a. acetylation b. phosphorylation c. methylation d. glycosylation

c. methylation

nucleotide units are linked together by ______________ bond to form nucleic acid a. hydrogen bond b. disulfide bond c. phosphodiester d. glycosidic bond

c. phosphodiester

whats lagging strand? a. the template used for RNA transcription b. the template for Okazaki fragment synthesis c. the new strand results from okazaki fragments ligation d. the DNA sequence have the similar sequence of RNA in transcription

c. the new strand results from okazaki fragments ligation

whats the right description on melting temperature? a. the melting temperature is the temerature that the dna was completly denatured b. in the measurement of melting temp with the increase of temperature the ABS260 decreases c. the single strand DNA have higher absorption at 260nm than double strand DNA d. the melting temp is the temp that dna and rna hybridize together with equal molarity

c. the single strand DNA have higher absorption at 260nm than double strand DNA

write the complementary sequence to the following template: 5' AGGTACTTAAGGCCTT 3' a. 5' AGGTACTTAAGGCCTT 3' b. 5' AGGTACTGGAGGCCTT 3' c. 5' TCCATGAATTCCGGAA 3' d. 5' AAGGCCTTAAGTACCT 3'

d. 5' AAGGCCTTAAGTACCT 3'

after DNA extraction you can perform a quick spectorphotometer reading at what wavelength do you measure the following DNA__________ RNA _____________ contaminants_______________

dna: 260 nm rna: 250 nm contaminents: 280 nm

what types of bonds are formed during hybridization when a strand of DNA finds its complement

hybridization is the formation of hydrogen bonds

You work in a lab and you have been asked to do a DNA extraction from bacterial cells. in one of the first steps you notice that you need to add the enzyme lysozyme. what is the purpose of adding this?

it breaks down the peptidoglycan cell wall

After a DNA extraction you can check the success of your extraction by either a spectrophotometer reading or running an agarose gel. list one reason why a spectrophotometer reading is more commonly done

it is much faster and cheaper to run

what is the purpose of doing a restriction digestion?

it is used in cloning in order to cut DNA to get a different gene to express what one gene has already expressed

if you want to detect the presence of multiple pathogens (eg. 3 total) in a single specimen what method would you choose

multiplex PCR

if you wanted to detect an RNA virus in a given specimen what method would you choose?

reverse transcriptase

HpaII cut site: 5'....C^CGG....3' 3'....GGC^C....5' does this restriction enzyme leave sticky or blunt ends

sticky ends

ddNTPs

terminator for sequencing, not naturally occurring

what determines the length of time required for step 3?

the size of amplicon determines the length of time for extension

why might the temp in step 2 be changed in different PCR runs?

the temperature depends and is adjusted based on the Tm of the primers (melting temperature)

compare and contrast PFGE with standard gel electrophoresis one similarity?

they both detect DNA band sized/ if run was successful

the following refer to PFGE: what is the purpose of this method

to detect large DNA fragments its used in DNA fingerprinting and identifying foodborn pathogens

In PCR reaction it takes a few cycles to generate the correct amplicon size. in the first cycle are the products too long or too short

too long- because it is stopped by a temperature change back to the 94 degrees c and the end is not reached

draw an example graph of data from a real-time pcr run. lable both axis and show the ct value on graph

y axis- concentration of flourescence x axis- PCR cycle # threshold line is important ct value-> value at wich it proceeds above the base line