Introduction to Molecular Diagnostics

molecular biology

the study of nucleic acids and molecular techniques used to handle and analyze DNA and RNA

DNA

molecule that contains instructions needed for an organism to develop, live, and reproduce

DNA is a macromolecule that contains these elements

PONCH

• phosphorus

• oxygen

• nitrogen

• carbon

• hydrogen

DNA is comprised of __________ in a _________ assembly

nucleotides; linear

two strands of DNA comprise—

the DNA helix

nucleotides

building blocks of DNA

nucleotide components

• 5 carbon sugar

• phosphate group carrying oxygen

• nitrogenous base

DNA and nucleotides are __________ charged

negatively

purines

adenine and guanine

pyrimidines

thymine, uracil, and cytosine

guanine forms __ _________ ____ with cytosine

3 hydrogen bonds

adenine forms __ _________ ____ with thymine (or uracil in RNA)

2 hydrogen bonds

changes in nucleotide structure

• methylation

• deamination

• addition

• substitutions

changes to nucleotide structure can be as a result of—

environmental factors like chemicals or radiation

changes to nucleotide structure can result in—

undesirable effects such as cancer

nucleic acid

macromolecule made up of nucleotides bound by phosphate and hydroxyl groups on their sugars

nucleic acid chains grow in the —

5’ to 3’ end by attaching the 5’ phosphate of a new nucleotide to the 3’ hydroxyl group of the end of a chain

the addition of nucleotides in the 5’ to 3’ way gives DNA its —

chain polarity

DNA replication begins at—

oriC

what enzymes unwind the double helix of DNA

topoisomerase and helicase

topoisomerase and helicase activity results in formation of —

a replication fork

primase enzyme

makes an RNA primer (6-11 bp)

DNA polymerase III

binds to the primer and adds DNA bases

leading strand

strand of DNA where DNA polymerase can continually bind and add bases

lagging strand (okazaki fragment)

primase repeatedly adds RNA primers in mall chunks followed by DNA polymerase

exonuclease

enzyme that cleaves RNA primers off the leading and lagging strands

DNA ligase

enzyme that seals strands to form double helix

DNA polymerase I

adds DNA bases to fill in lagging strand and removes RNA primers

DNA characteristics

• double stranded

• deoxyribose sugar

• thymine

RNA characteristics

• single stranded

• ribose sugar

• uracil

transcription

producing RNA copies of genes (DNA)

translation

production of proteins from mRNA (gene expression)

polyA tail

polyadenylic acid at 3’ terminus of mRNA strand

5’ cap

5’-'5’ pyrophosphate bridge to a methylated guanosine on 5’ end of mRNA

• protective function

• recognition site for translation machinery

DNA methylation

epigenetic regulation of gene expression found in C-G rich areas (CpG islands)

main mechanism of gene imprinting or gene silencing

DNA methylation

prader willi

example of genomic imprinting

• extreme feeding problems and delayed developement

• WT - paternal contribution of chr 15 expressed and maternal is silenced

• MUT - loss of paternal contribution

angelman’s

example of genomic imprinting

• seizures, speech difficulty, smiling, laughing demeanor

• WT - maternal UBE3A gene expressed and paternal is silenced

• MUT - deletion of maternal gene = no working copy of gene

MGMT stands for—

O6-methylguanine-DNA methyltransferase

MGMT is—

a DNA repair enzyme removing alkyl adducts from guanine

if MGMT gene promotor is silenced by methylation—

decreased MGMT enzyme production

prognostic biomarker for glioblastoma

MGMT promoter methylation

temozolomide

alkylating agent used to treat gliomas

• adding an alkyl group to guanine

• stop replication and induce apoptosis

MGMT positive tumor

MGMT pulls off alkyl group on guanine added by temozolomide

MGMT negative tumor

no production of enzyme, temozolomide can effectively add alkyl group to guanine

protein

polymer of amino acids

ultimate effect of nucleic acids is manifested in —

proteins

sequence of nucleotides on chromosome that encodes a specific functional product is—

the functional and fundamental unit of inheritance

triplet anture of nucleotide

3 nucleotide code using 4 nucleotide bases resulting in 64 possibilities that cover the 20 essential amino acids