1. Molecular Testing and Clinical Diagnosis
Molecular biology review
Part I
Slides 1-19 1

2. Objectives: At the end of the lesson the student should be able to:
Explain basic nucleic acid structure (DNA, mRNA, tRNA, rRNA), composition and function (C2)
Compare DNA and RNA similarities and differences (C3)
Explain and describe bonding pattern and specificity of the nitrogen bases (C2)
Locate of DNA and forms of RNA in prokaryotic and eukaryotic cells (C2)
Explain the processes of replication, transcription, and translation (C2)
Describe and evaluate target sequences for molecular testing (C3)

3. Cell diversity depends on:
Genome
genus species
composition
Gene expression
Protein products
structural
functional 3

4. Prokaryotes Simple unicellular organisms
bacteria (eubacteria, archaebacteria)
no internal organelles
circular strand of DNA
reproduce by fission 4

5. Gene control Allows single cells to adjust to nutritional environment
coupled transcription-
translation occurs
which decrease cell
cycle time
can take on external DNA through
Conjugation, transduction, transformation 5

6. Eukaryotic cells Contain internal organelles include:
membrane bound structures separate specific regions from the rest of the cytoplasm
include:
Fungi
Algae
Protozoa
plants
animals 6

7. Common organelles include
nucleus
smooth and rough endoplasmic reticulum
golgi
lysosomes, peroxisomes
some organisms have chloroplasts 7

8. Organelles Separate regions with unique function
nucleus
replication (DNA made into DNA)
transcription (DNA made into RNA
cytoplasm
translation (mRNA into Protein)
protect components from digestive enzymes in cytoplasm 8

9. Nucleic Acid Composition
Sugar
deoxyribose DNA
ribose RNA
Nucleotide base
Adenine, Guanine, Cytosine, Thymine
and in RNA, Uracil
Phosphate 9

10. DNA Structure Two strands of nucleic acid in opposite orientation
P-sugar backbone on the outside of ladder
nucleotide bases in the middle
hydrophobic interaction
P-sugar bonds are covalent
nucleotide base pair bonds are Hydrogen bonds
Note negative charge on P groups 10

11. bases are flipped or opposite
DNA strands are held precisely together in opposite orientation based upon sugar bonds involved in Phospho- di-ester bond
5` to 3` on one side
3` to 5` on the other side
bases are flipped or opposite 11

12. Opposite Orientation Allows:
Aligns nucleotide bases for hydrogen bonding
hydrogen bonding is :
a weak association between an electronegative atom (O or N) and a hydrogen atom
the hydrogen atom has an overall positive charge since its electron is utilized in the covalent bond 12

13. Nucleotides bind specifically
Adenine can only align and juxtaposition with thymine or uracil to form 2 hydrogen bonds
Cytosine can only align and juxtaposition with guanine to form 3 hydrogen bonds
Thermodynamically G-C pairs are stronger that A-T or A-U pairs due to the 3:2 hydrogen bonds 13

14. Overall Structure of DNA
Double helix
Opposite orientation
Held together by H bonds and hydrophobic interaction of nucleotidesC only
Inherent within the ACGT order of DNA is the specified protein product 14

16. Cell Cycle - G1, S, G2, M phases
G1 lag phase- cell growth
S phase- DNA is replicated prior to division (DNA to DNA: replication)
G2 lag phase- proteins are synthesized (DNA made into RNA: transcription and mRNA made into protein: translation)
M phase-mitosis cell division 15

17. DNA Replication-S Phase
DNA made into DNA: replication
Complex process requiring many enzymes, nucleosides bases and ATP
DNA must open up-helicase
DNA must stay open-ssDNA binding protein
DNA must be copied exactly-DNA polymerase 16

18. DNA polymerase Only adds nucleotides in the 5` to 3` direction
leading strand production-complimentary strand is produced in a continuous strand
lagging strand production-complimentary strand is produced in a discontinuous manner
Okazaki fragments
require RNA primers and ligase enzyme to link fragments together and remove RNA primers 17

19. synthesize DNA in the 5` to 3` direction Ligase
DNA polymerase
synthesize DNA in the 5` to 3` direction
Ligase
attaches two Okazaki fragments
5` P to 3` OH
Once DNA replication is complete cells enter G2 phase and prepare to divide by mitosis 18