Cell Bio Test 3

molecular cytogenetic technique used to detect chromosomal copy number variations (CNVs) across the entire genome with high resolution.

acgh

Double-stranded DNA is heated briefly to separate the two strands.

denaturation

The DNA is exposed specific primers— designed to bracket the region of DNA to be amplified—and the sample is then cooled to allow the primers to hybridize to complementary sequences in the two DNA strands.

annealing

This mixture is incubated with DNA polymerase and the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers. The process is then repeated by reheating the sample to separate the double-strand products of the previous cycle.

extension

PCR technique depends on the use of a special DNA polymerase

taq polymerase

“Taq Polymerase” isolated from a thermophilic bacterium

thermus aquaticus

• This polymerase is stable at much higher temperatures than eukaryotic DNA polymerases, so it is not denatured by the heat treatment

• The enzyme therefore does not have to be added again after each cycle

taq polymerase

What are the applications of PCR

forensics, genetics, mutagenesis

highly variable, short repeating DNA sequences (such as CACA... or GTGT...) located at specific positions (loci) throughout the human genome.

short tandem repeats

Why do unrelated individuals rarely share the same STR profile at a specific locus?

one from mom one from dad can span 4 to 40

Where do PCR primers bind when amplifying a specific STR locus?

non-repeating DNA sequences located on either side of the targeted STR locus

What does PCR produce when amplifying a single STR locus from an individual

pair of amplified DNA bands: one representing the maternal STR variant and the other representing the paternal STR variant

What determines the length and final position of an STR band during gel electrophoresis?

exact number of repeats at that locus. More repeats make the DNA fragment longer

What are the mathematical odds of two random individuals sharing the same DNA fingerprint when testing 5–10 STR loci?

1 in 10 billion

PCR can be used to obtain either

genomic or cdna clones

what are the ways to purify genomic dna

acgh, sequencing (identifying mutations), pcr (cloning)

what are the ways of purifying total rna

real time pcr, sequencing, rtpcr (cloning)

how to convert mrna to dna

hybridize with poly t primer, make dna with reverse transcriptase, degrade rna with rnase h, make complementary strand with dna polymerase

what can we study in endogenous genomic dna

mutations, gene organization, copy number

what can we study in endogenous rna transcripts

levels, splice forms, regulation

what can we study in endogenous proteins

levels, regulation, localization

to Add more gene

overexpress

to delete the gene

knockdown

to mutate the gene

site directed mutagenesis

what is the process for pcr making genomic clones

isolate total dna, seperate strands and add primers, pcr amplification

what is the process for pcr making cdna clones

isolate total mrna, add first primer, reverse transcriptase, and deoxyribonucleoside triphosphates, seperate strands and add second primer, pcr ampplification

what are the ways of isolating proteins

sds page, western blot (rrpa), localization (subcelluar fractionation)

• Quantitative high throughput method

• Femtogram sensitivity; nanograms of starting protein

• Requires high quality validated antibodies and robotics

• 200 antibodies validated

• Up to 1000 samples per slide

reverse phase protein array

• bacterial enzymes which cut at specific nucleotide sequences in the DNA.

• 4-8 bp

restriction nucleases

true or false: Each bacteria expresses a different restriction enzyme and these recognize a different sequence of DNA

true

Separates DNA Fragments of Different Sizes

gel electrophoresis

• a dye that intercalates into the DNA double helix and fluoresces under UV light

• gel electrophoresis

ethidium bromide

what are the analytical methods that use dna base pairing

southern blotting and cgh

what are the analytical methods that use rna base pairing

northern blotting, in situ hybridzation, microarrays

(1) Process of making many identical copies of a DNA sequence

(2) Begins with the isolation and manipulation of the DNA sequence

dna cloning

what happens in the isolation and manipulation of dna for cloning

restriction nucleases cut dna, two pieces are joined with ligase, can use a plasmid as a carrier to ligate this amplifies dna

DNA Cloning Begins with…

the production of recombinant dna

when there is a stagerred end that gets filled in for cloning what does that tell you about the restriction enzyme

the two fragments being joined were cut by different restriction nucleases

a staggered end in clning is filled in by

dna polymerases and dtnp

a vector contains…

promoter elements, origin of replication, antibiotic selection markers

recombinant dna are inserted into

plasmid vectors

true or false it is not the norm for plasmid dna to have staggered edges when cut

false

what is this process and what special feature does it grant the plasmids

bacterial transformation and antibiotic resistance

how are proteins expressed in large amounts

gene of interest cloned into expression vector with strong promoter

describe the process of getting overexpressed proteins

restriction nuclease cuts downstream promoter, protein-coding DNA coding sequence inserted, put it in cell

Conversion of signals that carry information from one form to another

signal transduction

the _______ signal molecule focuses on input while the _______ signal molecule focuses on output

extracelluar, intracellular

signaling system that controls hormones into the blood, extensive network

endocrine

signaling system that are local mediators

paracrine

signaling molecule that spans long distances but are directed (brain)

neuronal

signaling system that is dependent on cell to cell contact, no signal is actually released

contact dependent

self produced local mediators

autocrine

Each receptor type becomes activated by only ___ type of signal.

1

how do cells recognize a signal

receptors

• origin is adrenal gland

• hormone

• derivative of tyrosine

• increases blood pressure, heart rate, metabolism

epinephrine (adrenaline)

• origin is adrenal gland

• hormone

• steroid (cholesterol derivative)

• affects metabolism of proteins, carbohydrates, and lipids in most tissues

cortisol

• origin of ovary

• hormone

• steroid (cholesterol derivative)

• induces and maintains secondary female sexual charcteristics

estradiol

• origin is beta cells of pancreas

• protein

• hormone

• stimulates glucose uptake, protein synthesis, and lipid synthesis in various cell types

insulin

• origin is testis

• steroid (cholesterol derivative)

• hormone

• induces and maintains secondary male sexual charcteristics

testosterone

• origin is thyroid gland

• tyrosine derivative

• hormone

• stimulates metabolism in many cell types

thyroid hormone

• origin is plant tissues

• gaseous hydrocarbon

• hormone

• regulates developmental processes including seed germination and fruit ripening

ethylene

• origin is various cells

• protein

• local mediators

• stimulate epidermal and many other cell types to proliferate

epidermal growth factor (EGF)

• origin is various cells including blood platelets

• protein

• local mediators

• stimulates many types of cells to proliferate

platelet-derived growth factor (pdgf)

• origin various innervated tissues

• protein

• local mediators

• promotes survival and axonal growth of certain classes of neurons

nerve growth factor (ngf)

• origin mast cells

• local mediators

• histidine derivative

• causes blood vessels to dilate and become leaky, helping to causes inflammation

histamine

• local mediators

• origin is nerve cells and endothelial cells lining blood vessels

• dissolved gas

• causes smooth muscle cells to relax, regulates nerve cell activity

nitric oxide

• neurotransmitters

• origin is nerve terminals

• choline derivative

• excitatory neurotransmitter at many nerve-muscle synapses and in central nervous system

acetylcholine

• neurotransmitters

• glutamic acid derivative

• origin nerve terminals

• inhibitory neurotransmitter in central nervous system

y-aminobutyric acid (GABA)

• contact dependent signal molecules

• origin prospective neurons; various other developing cell types

• inhibits neighboring cells from becoming specialized in the same way as the signaling cell

• transmembrane protein

delta

intraceullar receptors are located on the…

cytosol or nucleus

ligands that bind to cell surface receptors

Large Hydrophilic Ligands

ligands that bind to intracelluar receptors

Small Hydrophobic Ligands

• origin many cell types

• protein

• inhibits cell proliferation, stimulate extracelluar matrix production

transforming growth factor (TGF-B)

what are the large hydrophilic ligands

egf, pdgf, tgf-b, ngf

what are the small hydrophobic ligands

cortisol

true or false: Same Signal can induce Difference Responses in Different Target Cells

true

• Signaling molecule

• Triggers conformational change in receptor that will influence cellular function

ligand