Molecular Techniques

Study of DNA, RNA, and proteins and their role in cell replication and the transmission of genetic information

Techniques implemented in order to study and/or manipulate DNA, RNA, or proteins Examples: - PCR - Hybridization

Human Identification, identify organisms, determine genetic disease, molecular oncology, etc.

Basic structure of life Contains all of info to make you Half from mom; half from dad Housed in the nucleus in the form of chromosomes

Organization: Base pairs (bp) are the basic sequences Wrapped around proteins called histones Histones make up nucleosomes Several nucleosomes compose chromatin Chromatin will make up a chromosome Human genome = 23 pairs of human chromosomes comprised of 3.2 billion nucleotides XX – female XY - male

Smallest component: Deoxyribonucleotide triphosphates Composed of a nitrogen base, a deoxyribose sugar and a triphosphate group

Pyrimidines – one ringed structures - Thymine - Cytosine Purines – two ringed structures - Adenine - Guanine

dNTP’s attach to one another via phosphodiester bonds forming a string of nucleotides Strong covalent bond between the 5’ carbon of one pentose sugar and the 3’ carbon of another pentose sugar Attachment can only occur at the 3’ end Attachment of nucleotide causes loss of two phosphates

DNA is double stranded A purine and a pyrimidine form a base pair via hydrogen bonds Cytosine – Guanine (3 hydrogen bonds) Thymine – Adenine (2 hydrogen bonds) Therefore, purines and pyrimidines will be in equal amounts C-G form a stronger bond and require more energy to be separated. Mismatching occurs when the laws of complementary base pairing are not met.

Complementary base pairing creates a double helix Sugar phosphate backbone (“sides of ladder”) Phosphodiester bonds Bases in the middle (“rungs”) Hydrogen bonds Protects unique base sequence DNA is chemically stable

DNA is negatively charged, acidic, and hydrophilic Bases are hydrophobic and protected in the interior Polarity due to orientation

5’ to 3’ direction from the phosphate terminal to the hydroxyl end Strands are referred to as complementary Relationship to one another is referred to as antiparallelism

RNA is the nucleic acid structure encoded by DNA Function To store and transmit “information” from the nucleus to the cytoplasm of the cell for protein synthesis

• RNA possesses uracil instead of thymine Both are pyrimidine ring bases Thymine is methylated uracil Sometimes thymine is referred to as methyluracil Methylation is energy $ Thymine in DNA prevents mutation Uracil is prone to forming bonds with other bases and deaminated cytosine looks like uracil RNA is single stranded but DNA is double stranded. RNA leaves nucleus but DNA does not. RNA’s sugar is ribose, DNA’s sugar is Deoxyribose

Different types of RNA: Pre-mRNA, mRNA, rRNA, tRNA, etc.

Transcription DNA to RNA by RNA polymerase Pre-mRNA to mRNA Introns removed by splicesomes only leaving exons 5’ guanine methylated cap 3’ poly A tail Leaves the nucleus

Translation RNA to protein mRNA (codon for amino acid), tRNA (anticodon and amino acid), and rRNA A and P site of ribosome > becomes amino acid chain with peptidyl transferase

Reverse transcriptase RNA to cDNA

A mutation is an alteration in a gene or chromosome that causes diversity (positive or negative) Affects the phenotype of the individual, and if it is a heritable change, can affect offspring Phenotype – observable trait Genotype - nucleotide sequence responsible for phenotype

In summary there are so many mutations that occur it is hard to separate cause and affect. (Me) From notes: Hard to narrow it down to one specific mutation! 400 identified mutations that cause PKU by altering the PAH gene 600 identified mutations that cause Cystic Fibrosis More than 2000 identified mutations that cause cancer susceptibility

Some possible causes of mutations include: Inheritance - mutations can be passed down to offspring Error in DNA replication - spontaneous mutation caused by DNA not being repaired Physical and chemical agents - mutagens affect DNA causing mutations

Polymorphism Variation in the DNA sequence that is present in 1-2% in the population Example: ABO Blood Groups Example: Sickle Cell Anemia Single base substitution → Glutamic acid (GAG) replaced by valine (GTG) = dysfunctional beta globin molecule in hemoglobin. Con: Patient suffers from anemia Pro: Malarial pathogens are unable to infect sickle cells

Affects a single or small group of genes Types include: Silent – nucleotide that does not change amino acid Conservative – amino acid replaced by biochemically similar amino acid Non-conservative – amino acid replaced by biochemically different amino acid Non-sense – premature termination of translation Frameshift – insertion or deletion of nucleotide that changes the whole message. Disasterous!

Affects structure of chromosome Translocations – exchange of genetic information between chromosomes Deletions - loss of chromosomal material Insertions - gain of chromosomal material Inversions - fragment of chromosome is removed, flipped, and reattached.