MLS 117 LABORATORY NUCLEIC ACID STRUCTURE

PIPETTING EXERCISES DNA

-Deoxyribonucleic acid
Micropipette -Sugar: Deoxyribose
-usual unit: microliters
-different capacities Nucleobases:
Adenine, Thymine & Cytosine Guanine
Parts:
1. Adjustment Knob RNA
-can be rotated -Ribonucleic acid
-adjusts the volume capacity Sugar: Ribose
Nucleobases:
2. Pipette Tip Adenine, Uracil & Cytosine Guanine
- small volumes- yellow pipette tip
- larger volumes (up until 1000 ul) - blue
pipette tip

Plunger Position:
1. 1st stop- aspirate
2. 2nd stop- dispense

CHOOSING A MICROPIPETTE:
50 ul sample- from stock solution -Purines are bigger; pyrimidines are smaller
150 ul diluent – distilled water - bonded by hydrogen bonds

Different micropipettes shown: Structure

-5-50 ul (YES; can be used)- yellow tip DNA
-2-20 ul (NOPE) -Composed of Nucleotides
-10-100 ul (NOPE) -Nucleotides are 3-unit molecules
-100-1000 ul (YES; can be used)- blue tip -(base, sugar and phosphate)
-base is attached to alpha carbon
-Phosphate- 5’ carbon

SOLUTION PREPARATION AND PH METER

-Video to be updated
RNA
-base, sugar and phosphate
-base is attached to alpha carbon
-Phosphate- 5’ carbon
DNA MUTATION
Mutations- errors in base sequence during DNA
replication
- Alteration in genetic composition
- Changes in amino acid sequence

2 types:
1. Point -one base is replaced with another
base
For example: CACGTA
If mutated: CACCTA
- Guanine is replaced with cytosine
- 1 triplet of bases or codon is affected

Subtypes:
A. Missense- codon that codes for a different
amino acid

Original: CGCTAT
If RNAismade: GCGAUA
GCG- Alanine
AUA- Isoleucine

If mutated: CGCAAT
If RNAismade:GCGUUA
GCG- Alanine
UUA- Leucine

B. Silent- AA is not affected as both mRNA

codons code for the same AA

Original: CGCTAT
RNA: GCGAUA
AA: GCG- alanine
AUA- isoleucine

If mutated: CGCTAG
RNA: GCGAUC
AA: GCG- alanine
AUC- Isoleucine
*Person affected will most likely not manifest a
condition
C. Nonsense- mutation results to stop codon DNA ISOLATION
- this will result to premature
termination of protein synthesis -routine procedure to collect DNA for subsequent
molecular or forensic analysis
Original: CGC AAT 3 steps:
RNA: GCGUUA A. Cell lysis
GCG: Alanine B. DNA precipitation
UUA- Isoleucine C. DNA purification

Mutated: CGCATT DNA analysis

RNA: GCGUAA -paternity -COVID testing
GCG- Alanine -forensic cases
UAA- Stop
A. Cell Lysis
*If there is nonsense mutation, the protein will be a • Physical methods: Blending, grinding, or
non-functional protein sonicating the sample.
-sonicating: involves producing ultrasonic
vibrations to break the cell membrane
2. Frameshift – inserts or deletes a base in a • Chemical method: Detergents and dissolved
sequence cellular proteins such as protease/ proteinase/
- Will affect all triplets of bases that peptidase.
follow
• The extraction of chromosomal DNA from
Original: CGA CGA TTA bacterial cells is a straightforward procedure. The
cells are broken up to release their content
If there is deletion(red) in 6th position from 5’: of DNA, RNA, proteins, and other components.
(start from the right or 5’)

CGA CGA TTA, (C is deleted) B. DNA precipitation

CGA GA TTA CGA GAT TA • The DNA can be precipitated out of the aqueous phase
by alcohol [ethanol] in the presence of a highly
*last triplet will not be coded due to deletion concentrated monovalent cations [e.g Na+].
*whole sequence is affected
Example: • This separates the DNA from other cellular debris by
neutralizing the negative charges of DNA molecules.
If there is insertion(red) of A in 6th position from 5’:
This involves stabilizing the DNA and making it less
water soluble with the use of NaCl solution.
CGA CGA TTA
CGC CAG ATTA (A is inserted) NaCl- to stabilize DNA

• Ice-cold ethanol or isopropanol is added to precipitate

the DNA molecules since the latter are insoluble in these
compounds.

• The precipitated DNA is dissolved in a buffer solution

containing EDTA [Ethylene Diamine Tetra Acetate],
which is a chelating agent to binds divalent cations.
DNA ISOLATION

• Since enzymes that degrade DNA [i.e endo and exo-

nucleases] need divalent cations, such as Mg+2, for their
activity, the presence of EDTA significantly decreases
DNA digestion.

-DNA should not be degraded by endo and exo

nucleases; hence the use of EDTA

C. DNA Purification

• The DNA can be purified from other debris by

extraction with phenol and chloroform. These are
organic solvents that dissolve and denature proteins,
dissolve lipids, and dissociate polysaccharides.

• This involves rinsing the DNA molecules with alcohol

to remove unwanted debris. The DNA then is dissolved
in water for easy storage and handling.

-Involves centrifugation