Molecular Diagnostics Chapter 1

trait

particular characteristic

Which elements make up the macromolecule of DNA?

carbon, nitrogen, oxygen, phosphorous, and hydrogen

What is the structure of DNA?

phosphorylated ribose sugar (deoxy ribose) backbone with a nitrogenous base (guanine, adenine, cytosine, thymine) two strands joined together through hydrogen bonds form a double helix

What type of bond joins two nucleotides?

phosphodiester bond

nucleotide

a nucleic acid with a phosphorylated ribose sugar and a nitrogen base, triphosphate

nuclein

viscous substance isolated from nuclei, discovered by Miescher and later found to be DNA

nitrogen base

a carbon-nitrogen ring structure that is part of a nucleoside

deoxyribose

ribose without a hydroxyl group on the carbon in position 2 on the sugar ring, with a hydroxyl group on the third carbon which is important for phosphodester bond formation

The. sequence is the ________ of nucleotides.

order

nucleoside

ribose sugar (unphosphorylated sugar) + nitrogenous base, adenosine, guanosine, cytidine, thymidine

purine

adenine, guanine (think Ag gold is pure, still has ring formation) double ring structure

pyrimidine

cytosine, uracil, thymine (think CUT, ring structure has been "cut") single-ring

Does adenine thymine pairing or cytosine guanine pairing have three hydrogen bonds?

cytosine + guanine (think c&g closer in alphabet, closer in space)

Which carbon on the deoxy ribose sugar forms a phosphodiester bond? Which catbon is the phosphate group attached to? What is this process called?

the third carbon, the fifth carbon, DNA polymerization

What is the difference between B-form, A-form, and Z-form DNA?

B- hydrated, 10.5 steps, right handed

A- dehydrated, 11 steps, right handed

Z- torsional stress, 12 steps, left handed

What is the directionality of DNA? What is the directionality of the complementary strand? What is the orientation of DNA?

5' to 3', 3' to 5', antiparallel

hybridization

formation of hydrogen bonds between two strands of DNA

Will single strands of DNA with identical sequences hybridize?

No, due to antiparallel orientation of DNA

denaturing agents

formamide, urea, mercaptoethanol, displace hydrogen bonds and separate two DNA strands

major groove and minor groove

two phosphate sugar backbones arranged as specific distances from each other, site of interaction with proteins or recognition sites

DNA synthesis occurs in which direction?

5'-->3'

DNA polymerase reads the template strand in which direction?

3' to 5'

What replication model describes the key to maintaining the sequence of nucleotides in DNA through new generations?

semi-conservative model of DNA replication

Does the leading strand or lagging strand have Okazaki fragments?

lagging strand because DNA polymerase reads in the 3'-->5' direction and the template strand is in the 5'-->3' orientation leaving jumps between sections of DNA replication

What is the orientation of the leading strand?

3' to 5'

DNA synthesis cannot proceed if a deoxyribose 3'hydroxyl oxygen is not available. Which enzyme provides the preceding base?

primase

What is the function of helicase?

unwinds the DNA double helix for replication

holoenzyme

polymerase II functions with a larger assembly of proteins to perform priming, initiation, regulation, and termination

replisome

complex of multiple proteins involved in replication

RNase H

the enzyme that helps to remove the RNA primer during DNA replication

DNA polymerase III

In charge of synthesizing nucleotides onto the leading end in the classic 5' to 3' direction.

DNA pol I

Removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides

pyrophosphorolysis

The reverse of a nucleotide polymerization reaction, in which pyrophosphate reacts with the 3'-nucleotide monophosphate of an oligonucleotide, releasing the corresponding nucleotide triphosphate.

Klenow fragment

large protein fragment of DNA pol I with 5' --> 3' polymerase activity, loses exonuclease activity

mutations

copying error in DNA replication that results in base changes

nick translation

the phenomenon in which DNA polymerase uses its 5' to 3' exonuclease activity to remove a region of DNA and at the same time replaces it with new DNA in the 3' to 5' direction. Occurs when there are errors in base pairing or can be used for labeling DNA products in experiments for hybridization analysis

DNA ligase

enzyme that chemically links DNA fragments together in DNA replication, Okazaki fragments, or nick translation

Pyrimidine dimers are caused by

ultraviolet light

Terminal transferase

a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules

hybrid vigor

the characteristic of offspring that are stronger than their parents - produced through crossbreeding

Gametes

a mature haploid male or female germ cell that is able to unite with another of the opposite sex in sexual reproduction to form a zygote.

endonuclease vs exonuclease

Endonuclease cleaves Phosphodiester bonds within the polymer, exonuclease cleaves phosphodiester bonds at the ends of the polymer

restriction enzymes

an enzyme produced chiefly by certain bacteria as a basic immune system, having the property of cleaving DNA molecules at or near a specific sequence of bases.

Type II restriction enzymes

used most frequently in the laboratory - do not have inherent methylation activity in the same molecule as the nuclease activity. Can leave sticky ends or blunt ends.

Are type II restriction enzymes found on eukaryotes?

No, prokaryotes only

Which restriction enzymes have methylation activity?

Type I, II, IV, IIG

What is the basis for paternity testing and specimen identity?

restriction enzyme fragment analysis

What is the advantage of blunt ends over sticky ends?

blunt ends can be joined together and do not have to match while sticky ends must match exactly in order to hybridize or converted to blunt ends through exonuclease activity or polymerase activity

CRISPR

an adaptive immune system for bacteria and archaea, clustered interspaced short palindromic repeats and case protein which recognizes foreign DNA and keeps record of previous infections

PAM

Protospacer Adjacent Motif, necessary sequence found adjacent to the protospacer in the target DNA, discriminates target from self

Oligonucleotides

segments of nucleic acid that are 50 nucleotides or less in length

Why is it important in DNA isolation techniques to eliminate or inactivate exonuclease and endonuclease activity?

because these enzymes degrade DNA

DNase I

enzyme that digests DNA

Why is DNA stored in TE buffer?

TE buffer chelates ions which are needed for nuclease activity, to stabilize DNA

topoisomerase

corrects "overwinding" ahead of replication forks by interconverting isomers or relax supertwised DNA

Gyrase

untangles DNA through ds breaks; separates linked rings of DNA (type II topoisomerases)

Methyltransferase

enzyme that methylates newly synthesized DNA strands and are involved in mismatched repair

What is the difference in methylation between prokaryotes and eukaryotes?

prokaryotes use methylation to distinguish between self and non-self, nearly all sites are methylated or hemimethyalted. Eukaryotes use methylation in specific regions to promote or prevent gene transcription

Recombination

the genetic process by which one chromosome breaks off and attaches to another chromosome during reproductive cell division (crossing over)

Conjugation

genetic recombination in bacteria F+ (fertility factor)-->F- bacteria which then becomes F+ through a filamentous bridge

Transduction

bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell by infecting bacterial cells

Transformation

(genetics) modification of a cell or bacterium by the uptake and incorporation of exogenous DNA, Griffith experiment, basis for recombination in labs

Protease

enzyme that digests protein

Ribonuclease

digests RNA

Why are plasmids more efficient at transformation compared to strands of DNA?

less likely to be degraded by physical shearing and chemical degradation during the process due to the circular shape

Episomes

plasmids that can integrate into the genome

acridine dyes

induce the loss of episomes

R plasmid

A bacterial plasmid carrying genes that confer resistance to certain antibiotics.

What is the original genetic material?

RNA

RNA

single-stranded nucleic acid that can fold upon itself or pair with complementary DNA/RNA to form a double helix, ribose sugar, uracil base

gene expression

conversion of the information encoded in a gene first into messenger RNA and then to a protein

Transcrption

The first step of protein synthesis, in which the information on mRNA is "read" and translated into a sequence of amino acids, the building blocks of a protein, occurs during interphase of cell division

RNA polymerase I,II, III

Pol I-rRNA (nucleolus) and III-tRNA (nucleus) synthesis noncoding RNA, Pol II (nucleus) synthesize mRNA

Promoter

specific region of a gene where RNA polymerase can bind and begin transcription

Does RNA synthesis require a primer?

No

Is DNA synthesis in prokaryotes or eukaryotes faster? What about RNA speed?

DNA 1000bp/sec vs. DNA 50 bp/s. DNa 50 vs RNA 50-100 bp/ sec

rRNA

ribosomal RNA; type of RNA that makes up part of the ribosome-the site of protein synthesis, most common type making up 80-90% of cellular RNA

mRNA

messenger RNA; type of RNA that carries instructions from DNA in the nucleus to the ribosome

polycistronic mRNA

molecules of mRNA that code for multiple proteins

Monocistronic mRNA

mRNA that codes forsingle type of protein, such as is found in eukaryotic cells.

constitutive transcription

state in which a gene is continuously transcribed

inducible/regulatory transcription

Transcription that is regulated by proteins and other factors

polyA tail

a sequence of 50-250 adenine nucleotides added onto the 3' end of a pre-mRNA molecule

polyadenylate polymerase

an enzyme that uses ATP to add 200 A's to the end of the polyadenylation sequence in eukaryotes on pre mRNA

mRNA capping

Addition of a 5' cap consisting of a 7-methylguanosine to the 5' end fo the primary transcript.

Does prokaryotic mRNA have introns?

No

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins.

snRNA

Found only in the nucleus of eukaryotes and functions to remove introns from mRNA (small nuclear RNA)

tRNA

An RNA molecule that functions as an interpreter between nucleic acid and protein language by picking up specific amino acids and recognizing the appropriate codons in the mRNA (transfer RNA)

RNA polymerase I, II, III locations,. njjnn

I-nucleus, II-nucleolus, III-cytoplasm

cDNA

Complementary DNA. DNA produced synthetically by reverse trascribing mRNA. Because of eukaryotic mRNA splicing, cDNA contains no introns.

What stereochemistry do proteins have?

L

What groups are attached to the central carbon of an amino acid?

hydrogen atom, amino group, carboxyl group, and a side chain

What are the types of amino acids?

polar, non-polar, aromatic, positively charge, negative charge

Zwitterion

A molecule that contains charges, but is neutral overall. Most often used to describe amino acids

What type of bond holds amino acids together?

peptide bonds

What type of reaction takes place during protein synthesis?

dehydration

zinc finger motif

A type of DNA-binding motif in regulatory proteins that incorporates zinc atoms in its structure.

leucine zipper

type of transcription factor in which repeating leucines form an interaction domain

conjugated proteins

Proteins requiring a covalently bonded prosthetic group to function properly. Hemoglobin is an example.