Molecular Testing and Clinical Diagnosis

Molecular biology reviewPart I

Slides 1-19

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)

Cell diversity depends on:

• Genome– genus species– composition

• Gene expression

• Protein products– structural– functional

Prokaryotes• Simple unicellular organisms

• bacteria (eubacteria, archaebacteria)

• no internal organelles

• circular strand of DNA

• reproduce by fission

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

Eukaryotic cells• Contain internal

organelles– membrane bound

structures separate specific regions from the rest of the cytoplasm

• include:– Fungi– Algae– Protozoa– plants– animals

Common organelles include

• nucleus

• smooth and rough endoplasmic reticulum

• golgi

• lysosomes, peroxisomes

• some organisms have chloroplasts

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

Nucleic Acid Composition

• Sugar– deoxyribose DNA– ribose RNA

• Nucleotide base– Adenine, Guanine,

Cytosine, Thymine – and in RNA, Uracil

• Phosphate

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

• 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

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

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

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

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

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

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

• 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