Molecular Diagnostics SLO

Module1: DNA, RNA, and Proteins

DNA

1. Describe the basic structure of nitrogen bases, nucleosides, and

nucleotides.

 Be able to number the carbons 1’ has the OH and H on it is a

pentose
 A nucleoside is when the sugar has the base attached to the 1’
carbon
 A nucleotide is when there is 3 phosphate’s are attached in a line
on the 5’ carbon

2. List and describe the 3 possible forms of DNA.

 B form is the most common, center of symmetry is in the helix,

right handed and 10.5 bp per turn

 A form is seen infrequently, center of symmetry is outside of the

helix, 11 bp/turn

 Z form is left handed, has extreme torsional stress and is 12

bp/turn

3. Describe the base pairing rules for DNA and what is meant by the
terms antiparallel arrangement and semiconservative replication.

 The helixes are antiparallel matches to one another, Adenine and

Thymine are pairs with two hydrogen bonds and Guanine and
Cytosine are pairs with three hydrogen bonds

 Semiconservative replication is when there is one “parent” DNA

and one synthesized DNA

 It is 5’ to 3’

4. List the name and functions of all major enzymes used during DNA
replication. Relate them to the process of replication.

 Polymerases where new DNA molecules are added to the

template strand

 Helicase (general term) Unwinds and helps keep DNA untangled

 o Two types Topoisomerases which relax super twisted DNA
and Gyrases which untangle DNA and separate linked rings
of DNA called concatamers.

 Rnase H removes the primer RNA laid down by the primase

 Primase lays down short RNA primases, creates lagging strand so

DNA polymerase has a site of action, Okazaki fragment

 Ligase repairs nicks in the DNA strand with the new strand

 Single Stranded Binding Protein (ssbp)

5. Differentiate between the eukaryotic and prokaryotic DNA

polymerases.

 Prokaryotic

o DNA Polymerase I: Used for recombination, repair and

replication

o DNA Polymerase II: Used for repair

o DNA Polymerase III: Main polymerizing enzyme

 Eukaryotic
o DNA Polymerase alpha: Found in the nucleus, most active,
chromosome replication
o DNA Polymerase beta: Found in the nucleus, DNA repair
o DNA Polymerase gamma: Found in the mitochondria, DNA
repair
o DNA Polymerase delta: 3’ to 5’ exonuclease activity, found
in bone marrow

6. Construct a complementary DNA sequence from a single strand of

DNA.

7. List the enzymes that modify DNA and state their specific functions.

 Restriction Enzymes recognize specific base sequences and

cause breaks or restrictions at the DNA at those sites

 Exonucleases do not work on closed circular DNA

8. List and describe the other DNA metabolizing enzymes.

 Type II Restriction Enzymes

 o Have bilateral symmetry and might have blunt or sticky
ends (helps join pieces of DNA

 Methyltransferases add methyl groups, typically on Adenine and

Cytosine
o Prokaryotic DNA is hemimethylated
o Eukaryotic DNA is methylated in specific regions

9. Briefly describe the function of a restriction enzyme and what their role
is in nature.

 They catalyze the cleavage of foreign DNA such as ones infected

by a virus

What I think might be important:

Processivity: Staying on the template longer

Fidelity: Faithful copying

Substrate Specificity: Affinity for altered nucleotides

RNA

1. Construct a complementary RNA sequence from a single strand of

DNA.

2. List and describe the three main types of RNA & Differentiate the
eukaryotic and prokaryotic RNA polymerases.

 rRNA- largest component of cellular RNA, structural and

functional component of the ribosome

o Prokaryotic rRNA: 70S in size

o Eukaryotic rRNA: 80S in size

 mRNA- initial connection between the DNA and RNA and the
primary polypeptide

o Prokaryotes: mRNA is synthesized and simultaneously

translated. Polycistronic (one mRNA codes for more than
one protein)

o Eukaryotes: processes are separate, they are separated by

nuclear membrane barrio and mRNA undergoes processing
before translation. Monocistronic (one mRNA codes for
one protein)
  tRNA- bring the amino acids to the growing poly peptide chain
during translation

3. List the enzymes that modify RNA and state their specific functions.

4. Explain the process of transcription.

 It is the conversion of DNA to RNA, it begins at

o Initiation- with a promoter(a specific sequence of bases

that calls for the start of transcription). Gets regulated by
Cis factors which are DNA sequences that act as a mark for
initiation and control of RNA synthesis and Trans factors
which are proteins that bind to the cis sequence and direct
assembly of the transcription complexes in the correct
order

o Elongation-

o Termination in prokaryotes it responds to protein levels and

in eukaryotes respond to a polyadenylation site

5. Categorize non-coding RNA and its mechanism of action.

6. Explain mRNA processing.

 Polyadenylation, it adds the polyA tail at the 3’ end it is not

coded for by the DNA sequence

 Capping, 5’ terminus has a 5’-5’ pyrophosphate bridge to a

methylated guanosine, this occurs after the initiation of
transcription it serves as protection and recognition

 Splicing, introns are intervening sequences of noncoding DNA,

this is where they get cut out what are translated are the exons

Proteins

1. Explain nucleic acid methylation and its effect on gene expression.

 It’s a silencer gene which means that it prevents a gene from

expressing itself, it can maintain balanced gene expression in
growth and embryonic development.

2. Explain the process of translation.

  In the ribosome a tRNA attaches to a mRNA and it has an amino
acid attached, as it moves along it begins to move along the
mRNA a peptide begins to form, a second AA tRNA attaches and
the first peptide leaves, as the second tRNA begins to form its
own peptide the original tRNA leaves the ribosome.

3. Identify post-translational gene modifications.

 It occurs in CpG islands usually found around exons, promoter

regions and the 3’ ends of genes.

4. Define epigenetics and list examples of epigenetic phenomena.

 Epigenetics is how we control translation

o Methylation silenced

 Tumor suppressor gene

o Phosphorylation gene activation

o Ubiquitination silencing

o Acetylation gene is active

Module 2: Nucleic Acid Extraction, Quality and Quantity

Assessment, and Chapter 4
Chapter 3, Nucleic Acid Extraction:

1. Identify the basic reagents needed for DNA isolation and describe their
function.

 Dish soap (cell lysis), meat tenderizer (precipitation of salts and

proteins), booze (isolation of DNA)

2. Explain one method for isolating nucleated cells in suspension.

 Inorganic method is usually called “salting out” referring to a low

pH and high salt ratio to precipitate proteins and leaving the DNA
in solution

3. Detail what preparation steps may be needed for tissue samples before
extraction.


 4. Explain why microorganisms may need additional isolation steps to
release nucleic acid and list three.

5. Differentiate organic and inorganic isolation.

6. Describe solid phase isolation.

 It is a silica based material that will selectively bind the DNA, it

lets other contaminating materials be washed out of the sample
and the DNA can be released from the silica, it uses like
magnetic balls to attach to the sperm DNA

Chapter 3, Quality and Quantity Assessment:

1. List the 4 methods used to assess DNA quantity and quality.

 Electrophoresis

 Spectrophotometry

 Fluorometry

 Microfluidics

2. Describe agarose composition and identify from where it is derived.

 Its derived from seaweed

3. Explain how to prepare and run agarose gel electrophoresis. Include a

description of how/why DNA moves through the gel, what effects the
rate of movement, and how nucleic acids are visualized.

 DNA is negatively charged and goes to the opposite pole in

electrophoresis

 The more charged and smaller the molecule is the further it will
go in the gel

 These acids are visualized through EtBr or SYBR Green UV

additives

4. Diagram the relative positions of different DNA forms in agarose gel.

5. Assess agarose gels for quality of plasmid DNA, HMW chromosomal

DNA, and RNA AND quantity of DNA.
 6. Identify the wavelength at which DNA most strongly absorbs UV light
and explain why this is.

7. Identify the µg/mL equivalent of DNA and RNA for one OD unit.

8. Calculate the concentration of DNA given spec readings and dilution

information.

9. Identify why too much or too little DNA can negatively impact a
molecular assay.

10. List the wavelengths used to assess quality of DNA and the
reason each was chosen. Identify the ideal 260:280 ratio and what
values outside of this range, both high and low, can be caused by.

11. Assess quality of DNA using provided data from a

spectrophotometer.

Chapter 4:

1. Identify where agarose is derived from.

 It’s a polysaccharide polymer from seaweed, it is nonpolar

2. Calculate how to make agarose gels in the usual percentages for DNA
and RNA work.

 It is the percentage provided divided by 100 the multiplied by

the mL needed

3. Explain the reason to use a DNA ladder (MW marker) and a loading
(tracking) dye.

 It provides a key for the dyes after electrophoresis is completed

4. Describe the purpose of buffers for AGE and compare and contrast TAE
and TBE.

 It resists the changes in pH

o TAE is very quick and is preferable

o TBE is slow and low, better for precise separation and it

does not store well

5. List 4 buffer additives and explain why each would be used.


 6. Explain why RNA will require gel or buffer additives and list what they
are.

7. Discuss the relative safety of ethidium bromide compared to SYBR

green and explain why these chemicals are needed for AGE.

 They make DNA fragments visible after electrophoresis, EthBr is

a mutagen and a powder and very dangerous to handle, more
specifically inhale or ingest. SYBR Green is still a mutagen and
still requires UV light like EthBr but it reduces the concentration
and reduces safety risks and it is 25-100x more sensitive than
EthBr

8. Estimate the size of DNA fragments in an agarose gel and evaluate it

for anything that went wrong.

Module 3: Analysis & Characterization of Nucleic Acids

and Probes
1. Explain why restriction enzyme mapping is used and how it can be
applied to monitoring base changes.

2. List the macromolecules detected for Southern, Northern, and Western

blotting.

3. List the main steps, in order, of the Southern Blotting procedure.

4. Evaluate a gel run after restriction enzyme digestion to determine if it

was successful and troubleshoot issues, if needed.

5. Explain why DNA needs to be depurinated and denatured before

transferring to a membrane and describe how this is done.

6. List 3 ways DNA fragments may be transferred from a gel to a

nitrocellulose membrane and provide a one sentence description of
how each works.

7. Summarize the reason for the prehybridization step and provide and
example of a reagent that may be used.

8. Briefly describe what a probe is and list 3-4 types of detection

molecules you can use.

9. Describe stringency and describe 3 ways it can be increased or

decreased.
10. Summarize how a protein "probe" differs from nucleic acid
probes.

11. Evaluate Southern and Northern blot data and draw conclusion
from those results.

12. Evaluate Western blot data and draw conclusions from those
results.

13. Summarize the main difference between dot and slot blots and
how they differ from Southern, Northern, and Western blots.

14. Evaluate dot and slot blot data and draw conclusion from those
results.

15. Describe the major benefit to macroarrays and microarrays and

briefly summarize how the two differ from each other.

16. Evaluate macro- and microarray data and draw conclusions from
those results.