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

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 3

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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