question

Pyrimidine

answer

one carbon ring Cytosine, Thymine, Uracil

question

Purine

answer

two carbon rings Adenine, Guanine

question

How are nucleotides joined together?

answer

condensation

question

What does mRNA do?

answer

carries code out of cell. code for protein structure. DNA to ribosomes end of transcription start of translation

question

What does tRNA do?

answer

decodes mRNA. Matches amino acids to mRNA code. Protein synthesis

question

What does rRNA do?

answer

part of ribosome structure most abundant RNA coordinated coupling of tRNA to mRNA codons

question

Feedback Inhibition

answer

end product of pathway is noncompetitive inhibitor binds to allosteric site to slow down rxn b/c too much product

question

Exonucleases

answer

degrades nucleic acids by removing one terminal nucleotide at a time cleaves phosphodiester bond at end of chain 5' ---> 3' and 3' ---> 5'

question

Endonucleases

answer

cleaves phoshpodiester bonds w/in chain recognition site is palindromic sequence cleaves unevenly, leaves sticky ends (restriction enzymes)

question

DNA polymerase III

answer

copies DNA by reading existing strand, builds new comp. strand always adds to 3' end can't start new strand on its own (ORI sites)

question

ORI sites

answer

origin of replication on chromosome triggers DNA replication

question

gyrase

answer

unwinds DNA in front of fork

question

DNA polymerase I

answer

removes RNA nucleotides form RNA primers and replaces with DNA (makes sure primers that start replication are removed)

question

helicase

answer

breaks H-bonds of double helix

question

topoisomerase

answer

breaks/ reseals DNA backbone to release tension caused by twisting (DNA gyrase)

question

primase

answer

(RNA pol) primers, small RNA, serves as starting points for copying DNA helper enzyme for DNA pol III

question

single-strand DNA-binding proteins (SSBPs)

answer

hold DNA open during replication

question

Okazaki fragments

answer

lagging strand 3' ---> 5' away from fork DNA pol III adds, DNA ligase closes gaps

question

Ligase

answer

closes gaps in DNA forms covalent bonds b/w nucleotides, 3' end of new DNA to 5' end of growing chain

question

What are the steps in DNA replication?

answer

1. Initiate 2. Elongate 3. Terminate

question

Telomers

answer

cap ends of chromosome during replication to preserve DNA

question

-10 box

answer

TATA box (TATAAT) located 10 bases away from transcription start site promoter RNA pol binding site

question

-35 box

answer

TTGACA located 35 bases away from transcription start site promoter

question

RNA Polymerase

answer

opens double helix reads one strand to make RNA, 3' --> 5' (antiparallel) DNA-dependant catalyzes formation of covalent bonds b/w nucleotide and growing chain

question

splicesomes

answer

made up of snRPs remove ntrons during transcription cut out intron and reform bonds b/w exons

question

enhancers

answer

far from promoter maintain a tissue-specific or cell-specific level of gene expression

question

Poly-A tail

answer

keeps mRNA from being destroyed in cytoplasm at 3' end piece of 100-250 A's

question

5' cap

answer

added to 5' end of finished mRNA let's cell know it's ready for translation ribosomal binding site

question

aminoacyl tRNA

answer

tRNAs that carry amino acids

question

Ribosomes

answer

where mRNAs and tRNAs meet Large and small form packets (A, P and E) catalyzes peptide bond formation b/w amino acids by rRNA

question

What is the path of a tRNA in a ribosome?

answer

A ---> P ------> E

question

How is translation initiated?

answer

small subunit binds to ribosomal binding site MET anticodon binds to start codon large subunit binds to small subunit

question

How is translation terminated?

answer

stop codon is carried into small subunit by tRNA recognized by release factor, breaks bond b/w chain and tRNA released from ribosome and out into cell

question

reverse transcriptase

answer

enzyme that allows RNA to go to DNA mRNA --> ssDNA --> cDNA --> dsDNA

question

Pleiotrophy

answer

a single gene controls the expression of many phenotypic traits ie Sickle Cell Anemia

question

cDNA

answer

intron free complementary DNA (mRNA already spliced it) can be inserted into a plamid

question

Vector

answer

helps carry DNA into cell ie plasmids, virus

question

Exons

answer

protein info this is what is translated separated by introns

question

Open Reading Frame

answer

sections of DNA that begin with start codons and end with stop codons DNA: 5' --> 3' transcription: 3' --> 5' DNA --> RNA (promoter) translation: 5' --> 3' mRNA

question

Mass Spec

answer

quantitate measures amount of light passing thru

question

What is the wavelength of RNA or DNA in mass spec?

answer

260 nm

question

What is the wavelength of protein in mass spec?

answer

280 nm

question

Steps in liquid phase DNA isolation

answer

1. Lyse 2. Separate out contaminates (by spinning) 3. Add phenol/ chloroform. Mix and separate. Remove and keep upper layer 4. Precipitate out DNA w COLD EtOH 5. Treat to remove RNA 6. Inactivate treatment 7. Deproteinization with phenol 8. Re-precip DNA with EtOH

question

How do you inactivate RNases?

answer

200 degrees for 2hrs 30 min in 1M NaOH or quanidinum isothiocyanate

question

How much restriction enzyme do you use for a DNA digest?

answer

10 U of enzyme per ug of DNA

question

Hybridization

answer

2 single-stranded DNA molecules of comp base sequence can form a double-stranded hybrid (duplex)

question

What does the incubation step in hbridization do?

answer

Allows formation of double-stranded molecules

question

Blocking DNA (Hybridization)

answer

minimizes probe binding to nonspecific sequence ie salmon sperm DNA, Human LINE-1

question

Blocking Proteins (Hybridization)

answer

minimize nonspecific binding of probe to membrane ie casein (milk), Denhardt's sol

question

Stringency

answer

conditions of hybridization that control the specificity of binding b/w the probe and target sequence

question

How can you increase strigency in a hybridization?

answer

increase temp decrease salt conc.

question

Formanide acts as a __________ in a hybridization.

answer

denaturing agent

question

Line Probe Assay

answer

LiPA reverse hybridization assay using sequence-specific oligonucleotide probes (reverse SSOP) multi-parameter testing --> single strip

question

Line Probe Assay steps

answer

1. Isolate nucleic acid (RNA) 2. Amplify 3. Hybridization 4. Strigent wash 5. Conjugate incubation 6. Substrate detection

question

What method would you use if you knew the gene sequence and the mutation?

answer

Reverse Dot Blot

question

Microarrays

answer

Used for unknown gene and mutation cDNA libraries can be used for gene expression, tumors, genetic mapping, mutations and polymorphism large scale, high throughput analysis

question

Microarray steps

answer

1. get mRNA from cells 2. RT to get labeled cDNA copies of mRNA 3. cDNA washed over slide. cDNA sticks to comp sequence 4. use lser to read fluorescent tags

question

Southern Blot

answer

can detect a specific DNA fragment among many

question

What can Southern Blots be used to detect?

answer

deletions/ insertions point mutations polymorphisms structural rearrangements

question

Southern Blot steps

answer

1. Restriction enzymes cut DNA into fragments 2. Run on gel to separate 3. Soak gel in alkali to denature dsDNA 4. Transfer ssDNA fragments to positively charged membrane (blot) 5. Fix to filter by heat (80*) or UV crosslink 6. Incubate (hybridize) blot w/ radioactively labeled ssDNA comp probe 7. Autoradiograph

question

Heterduplex Analysis (HA)

answer

use for known gene, unknown mutation mutation screening bands on gel --> retarded migration from WT due to seq differences

question

Heterduplex Analysis steps

answer

1. PCR 2. Mix sample and CTR DNA together 3. Denature PCR using heat 4. Cool slowly to rt 5. Add denaturing loading buffer 6. Run on MDE gel

question

SIngle-Stranded Conformational Polymorphism Ananlysis (SSCP)

answer

used for known gene, unknown mut mutation screening short PCR products takes 3D shape when cooled --> muts have different shape than WT non-denaturing PAGE

question

What makes DNA negatively charged?

answer

all the phosphate groups

question

How does EtBr cause DNA to fluorese?

answer

intercalates into the double helix

question

TAE Buffer

answer

tris-acetate w/ EDTA good for DNA recovery good for lg fragments low buffering capacity increases migration of DNA thru gel

question

TBE Buffer

answer

tris-borate w/ EDTA good for small DNA fragments high buffering capacity decreases migration thru gel

question

Pulse Field Gel Electrophoresis steps

answer

1. culture 2. embed pellet in agarose plug 3. treat w/ lysozyme (cell lysis) 4. proteinase K 5. restriction enzyme digest 6. gel

question

How can Tween 20 affect PCR?

answer

stablizes Taq supress formation of 2* structures increase yeild increase non-specific amplification

question

What are some disadvantages of PCR?

answer

must know the sequence first highly contamiable may not be 100% specific specificity dependant on temp and Mg++ conc

question

What is the purpose of primers in PCR?

answer

to intiate replication

question

What are the 3 main steps of PCR and their tempuratures?

answer

Denature 92* Anneal 50* Extenstion 72*

question

PCR process

answer

1. Mix: taq, primers, dNTPs, DNA 2. Separate dsDNA (Denature) 3. Cool. Allow primers to bind, taq to add nt to 3' end of primers (Anneal) 4. Denature dsDNA again (new copy and original) 5. Cool 6. Repeat steps 4-5

question

What can cause primer dimers?

answer

annealing temp too low too much primer

question

What are primer dimers?

answer

size is sum of two primers taq extends one primer, which is annealed to another primer

question

What can inhibit PCR amplification?

answer

detergent (SDS) phenol (left over from DNA isolation) herapin (specimen tube) heme dyes CSF, urine, sputum, parafilm

question

What is the use of uracil-N-glycosylase (UNG) in PCR?

answer

can help with contamination by deestroying dUTP (hich is used for RNA and not DNA

question

What are some causes of too many bands on a gel after PCR?

answer

primers not specific enough annealing temp too low too many cycles too much Mg++

question

RT-PCR

answer

RNA ---> cDNA first strand synthesis primer dependant polymerase dependant rxn

question

Methylated Sensitive PCR steps

answer

1. convert DNA w/ sodium bisulfate 2. clean DNA 3. Mutiplex PCR 4. analyze size of fragments on gel (size dependant on methylation

question

How does PCR work with methylated DNA?

answer

primers are designed to recognize methylated and unmethylated sense strands at gene promoter the methylated bases inhibit enzyme activity at recognition sites

question

Ligase Chain Reaction (LCR)

answer

amplification of copy number good for specific mutations uses DNA ligase two primers directed against target sequence

question

Which pathogens can LCR be used to detect?

answer

Chlamydia Gonorrhea Listeria HPV

question

Nucleic Acid Sequence Based Amplification (NASBA)

answer

isothermal rxn --> no thermocycler great sensitivity enzymatic rxns take place concurrently

question

NASBA Steps

answer

1. Hybridize oligo-T7P primer to target seq 2. RT 3. Digest with RNase H 4. Hybridize with target-specific oligo primer (P2) 5. RNA transcript of T7 RNA pol

question

What pathogens can you detect using NASBA?

answer

HIV hepatitis HTLV CMV

question

Transcription-Mediated Amplification (TMA)

answer

two enzymes: RT and RNA pol isothermic RNA or DNA

question

Strand Displacement Amplification (SDA)

answer

simultaneous amplification and real-time detection high throughput high sensitivity done within a 96-well plate

question

Branched DNA (bDNA)

answer

signal-dependant amplification instead of product-dependant no enzyme detection by signal from "capture probes" microtiter kinda like a hybridization

question

What are the clinical uses for DNA sequencing?

answer

mutation detection confirm mutation by other method resistance testing HLA genotyping

question

Do you have to know the gene sequence in orde to do DNA sequencing?

answer

yes, but you do NOT need to know the mutation

question

Sanger Sequencing method

answer

DNA obtained by res enzymes divided into 4 samples (A, T, G, C) label with radioactive oligo at 3' end mix with taq, dNTPs, ddNTPs and incubate run on gel --> 3' ends will differ by one base

question

Why does Sanger sequencing use ddNTPs instead of dNTPs?

answer

the lack of a 3' OH on the ddNTP makes it impossible for pol to add more nucleotides --> chain termination

question

ddNTP

answer

dideoxyribonucleoside triphosphate lack a hydroxyl group (OH) at 2' and 3'

question

Fluorescent in situ Hybridization (FISH)

answer

Does not require actively dividing cells known gene seq, unknown mut

question

What are some of the benifits of FISH?

answer

don't need mitotic cells ---> shorter TAT large number of cells may be scored dual color ---> mutiple targets many sample types

question

What kind of probes are used for FISH?

answer

painting (all or part of chromosome) alpha satellite (centromeres) single copy (specific regions on chromosome)

question

What is the wavelength for background on a mass spec?

answer

320 nm

question

How do you determine quality of DNA/ RNA using a mass spec?

answer

A260 - A320 A280- A320

question

What is considered good quality DNA/ RNA from a mass spec?

answer

1.7 - 2.0

question

What is considered poor quality RNA/ DNA from a mass spec?

answer

<1.7

question

What does smearing on a gel indicate?

answer

degredation loaded too much

question

How can you tell if you have good RNA using gel electrophoresis?

answer

good RNA will have a 2:1 intensity (28S : 18S) if 18S is more, RNA degradation is possible

question

What is one way you can increase the yeild / quality of DNA or RNA after running on a gel?

answer

Do an EtOH precip

question

Which specimen tubes are the best for use with molecular assay?

answer

EDTA (purple) can also use ACD (yellow)

question

Which specimen tubes are not good for use with molecular assays?

answer

Heparin (green) inhibits several enzymes used in molecular assays

question

Should you freeze blood or bone marrow if you are going to use it in a molecular assay?

answer

NO

question

What is the best temperature to store DNA?

answer

Long term = -70* short term = -20*

question

For long term storage, what should you store DNA in?

answer

EtOH

question

What is the best temperature to store RNA?

answer

Long term = -70* short term = -20*

question

Which is the starting template for PCR?

answer

DNA

question

Which is the starting template for RT-PCR?

answer

RNA

question

Should you lyse RBCs before freezing?

answer

YES

question

After an extraction/ isolation, what should you elute with?

answer

DNA - TE or water RNA - DEPC water

question

what are the to types of isolation/ extraction methods?

answer

liquid phase (phenol/ choroform) solid phase (qiagen)

question

Steps in solid phase DNA isolation

answer

1. lyse cells (lysis buffer) 2. denature/ digest proteins (proteinase K) 3. separate DNA from junk 4. precipitate DNA (wash 1 & 2) 5. elute off filter (elution buffer)

question

What some causes of DNA damage?

answer

mutagens carcinogens cell death age-related decreases in DNA repair genetic disease

question

Nucleotide Excision repair

answer

done by endonucleases and exonucleases

question

Base Excision repair

answer

done by DNA glycosylase, AP endonuclease, exonuclease

question

Methyltransferase repair

answer

transfers methyl group to itself from guanine

question

Direct Insertion repair

answer

purine insertase

question

How does DNA repair work in replication?

answer

if the damage is post replication, skips over damage, leaves gap and repair later

question

Diagnostic sensitivity

answer

likelihood of positives

question

Diagnostic specificity

answer

likelihood of negatives

question

Clinical sensitivity and specificity is based on _________.

answer

outcome

question

Analytical sensitivity is based on _____ and specificity is based on _______.

answer

limit of detection target specificity

question

What is direct analysis?

answer

identifying a specific gene or mutation that caues disease. gene must be known, might need to know mut

question

What are some direct analysis methods?

answer

southern PCR - ASO blot, restriction digest SSCP HA

question

What some indirect analysis methods?

answer

RFLP linkage analysis

question

What is indirect analysis?

answer

inherited marker near gene associated with disease unknown gene and /or mut

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