Fichas de aprendizaje ASCP Molecular Biology Certification Exam | Quizlet

Pyrimidine

One carbon ring
Cytosine, Thymine, Uracil

What is the function of mRNA?

Carries genetic info out of nucleus
Transcript translated to protein

What is the function of tRNA?

Carries aa to ribosome
Anticodon pairs with codon on mRNA strand

What is the function of rRNA?

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

Feedback inhibition

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

Endonucleases (Prok)

Restriction enzymes
Cleaves phoshpodiester bonds w/i poly-nt chain
Recognition site is palindromic sequence
Types I-V

ORI sites

nt sequence where replication is initiated

Topoisomerase I

Induces ss breaks
Remove DNA supercoils during TXN and DNA replication; for strand breakage during recombination; for chr condensation; and to disentangle intertwined DNA during mitosis

topoisomerase II

cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strands

Gyrase (topoisomerase II)

Unwinds supercoiling caused by unwinding at the rep fork by introducing DSBs

Helicase

Breaks H-bonds of double helix at the replication fork

single-strand DNA binding proteins (SSBPs)

Binds ssDNA and prevents it from re-annealing during TXN, replication, repair, and recombination

Telomeres

Repeat sequence (TTAGGG) at the ends of chr, protect chr from degradation

RNA polymerase

DNA dependent RNApol
Transcribes DNA template to RNA (3'-->5'; anti-parallel)

Splicesomes

Complex of snRNPs
Removes introns from pre-mRNA and splices exons together

Enhancers

Short regions of DNA that bind proteins (TXN factors) that enhance TXN of a gene

Reverse transcriptase

enzyme that transcribes RNA to cDNA (lacks introns)
RNA --> RNA:DNA --> cDNA (dsDNA)

Open Reading Frame (ORF)

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

Spectrophotometer

Measures amount of light absorbed
Quantitative measurement of [DNA/RNA]

At what wavelength does DNA and RNA absorb?

260 nm

At what wavelength does protein absorb?

280 nm

Organic isolation method

1. Lyse
2. Add phenol/ chloroform > vortex/spin
3. Transfer aqueous layer (top) to new tube
4. Add chloroform:IAA (removes phenol) > vortex/spin
5. Transfer aqueous layer to new tube
6. Add NaOAc and EtOH > vortex/spin
7. Decant
8. Resuspend

What does the incubation step in hybridization do?

Allows formation of ds molecules

Formamide acts as a __________ in a hybridization.

denaturing agent

Line Probe Assay (LiPA)

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

Single-Stranded Conformational Polymorphism Ananlysis (SSCP)

Used for known gene, unknown mut
Mutation screening
Short PCR products form 3D conformation when cooled --> muts have different conformation than WT
Non-denaturing PAGE, muts migrate different than WT

How does EtBr cause DNA to fluoresce?

Intercalates into the double helix
Absorbs UV ~300 nm, emits ~600 nm

Pulse Field Gel Electrophoresis steps

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

What are the 3 steps of PCR and their temperatures?

Denature 90-96C
Anneal 50-70C
Extension 68-75C

Bisulfite DNA sequencing/Methylation specific

1. RE digest
2. Electrophorese and purify fragment of interest
3. Denature and incubate w/ sodium bisulfate (turns C>U, methylated C is unchanged)
4. clean, ppt, and resuspend
5. PCR --> sequence
6. Compare treated vs untreated, note where CG are not changed to TA

NASBA steps

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

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

Yes, in order to design primers
You do NOT need to know the mutation

Sanger sequencing method

Divided into 4 samples (ddA, T, G, C)
Label with radioactive/dye oligo at 3' end
Mix with taq, dNTPs, ddNTP and incubate
run on gel --> frags will terminate at different lengths

Fluorescent in situ hybridization (FISH)

Uses fluorescent probes to detect DNA sequences on chr

What types of probes are used for FISH?

Dual fusion: 2 probes flank the breakpoint at both t locations
CEN probes: centromeric probes bind to repetitive alpha satellite sequences
Telomeric probes
Whole chr paints

What is the wavelength for background in spectrophotometery?

320 nm

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

A260/A280

What is considered good quality DNA/RNA from spectrophotometry?

DNA: 1.7-2.0
RNA: 2.0-2.3

What is considered poor quality RNA/DNA from spectrophotometry?

<1.7 Protein contamination

What is one way you can increase the yield/quality of DNA/RNA after running gel?

Do an EtOH ppt

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

DNA - TE or water
RNA - DEPC water

What are the two types of isolation/extraction methods?

Liquid phase (organic & inorganic)
Solid phase (Qiagen)

Nucleotide Excision repair (NER)

Done by endonucleases
Removes a span of nt's by cleaving phosphodiester bond

Base Excision repair (BER)

Done by DNA glycosylase, AP endonuclease
Cleaves glycosidic bond of a single base base, leaving apurinic/apyrimidinc site

Describe the growth of the nucleic acid chain

The chain grows by the attachment of the 5' phosphate group of an incoming nucleotide to the 3' hydroxyl group of the last nucleotide on the growing chain

Denaturing agents

formamide, urea, mercaptoethanol

Solenoid

a coil of six nucleosomes wound into a tightly packed helix

Mutation

DNA sequence change that is present in a relatively small proportion of the population <1%, somatic changes

Variant

inherited sequence alterations

Polymorphism

a change in the DNA sequence that is present in at least 1-2% of the population (ex. Sickle cell anemia)

Gene mutations

affect single genes and are often small changes in the DNA sequence

Chromosome mutations

Affects the structures of the entire chr, requires the movement of large chr regions

Genome mutations

Change in the number of chr's

Eupliod

normal complement of chromosomes

Aneuploid

Increased number of chr's (eg. Down's syndrome)

Haploid

Single copy of each chr (humans have 23)

Diploid

Two copies of each chr (humans have 46)

DNA polymerase

Catalyzes phosphodiester bond between nt's
Uses ssDNA as a template to determine which nt's to add

DNA Polymerase I (Prok)

Processes Okazaki fragments
Replaces RNA primers with DNA (exonuclease activity)
Excision repair & proof reading

DNA Polymerase II (Prok)

DNA repair, exonuclease activity

DNA Polymerase III (Prok)

Primary enzyme involved in replication
Exonuclease activity

DNA Polymerase IV (Prok)

Bypass replication
SOS response

DNA Polymerase V (Prok)

Bypass replication
SOS response
Translesion synthesis DNA repair

DNA Polymerase α (Euk)

Primase
DNA dependent DNA & RNA pol

DNA Polymerase β (Euk)

Base excision repair (BER)

DNA Polymerase δ (Euk)

Lagging strand synthesis
DNA repair, exonuclease, replaces primers as it encounters Okazaki fragments

DNA Polymerase ε (Euk)

Leading strand synthesis
exonuclease

DNA Polymerase γ (Euk)

mtDNA replication and repair
Exonuclease activity

Terminal transferase

DNApol synthesizes poly-nt chain at 3' end w/o a template

Transcription

initiation --> elongation --> termination

Retrotransposons

Mobile genetic elements which can increase genome size and insert itself within coding/noncoding regions

The three biochemical activities of reverse transcription

RNA-dependent DNApol, Ribonuclease H, and DNA-dependent DNApol --> all used to create ds cDNA from RNA

Describe the steps of reverse transctiption

1. tRNA acts as a primer and hybridizes to virus genome
2. Complementary DNA then binds to the U5 (non-coding region) and R region
3. RNAse H degrades the 5' end of the RNA which removes the U5 and R region.
4. The primer then "jumps" to the 3' end of the viral genome and the newly synthesized DNA strands hybrid

Splicing

modification of the nascent pre-messenger RNA (pre-mRNA) transcript in which introns are removed and exons are joined.

R-factors

resistance transfer factors. Carry antibiotic resistance to common antibiotics.

Colicinogenic factors

resistance to bacteriocins, toxic proteins manufactured by bacteria.

Primary protein structure

sequence of a chain of amino acids

Secondary protein structure

Amino acids are linked by H bonds to form sub structures; eg a helixes, B sheets

Tertiary protein structure

3D structure of a single protein

Quaternary protein structure

3D structure of a multi sub unit protein

Restriction enzymes

endonucleases that recognize specific sequences and break the phosphodiester bond of dsDNA

Type I restriction enzymes

have both nuclease and methylase activity in a single enzyme. Bind to host-specific DNA that contains methylated adenines

Type III restriction enzymes

resemble type I enzymes in their ability to methylate and restrict (cut) DNA. - adenine methylation occurs on only one strand.

Type II restriction enzymes

used most frequently in the laboratory - do not have inherent methylation activity in the same molecule as the nuclease activity

Exonuclease I

Degrades ssDNA from 3'-->5'

Exonuclease III

Removes 5' mono-nt's from the 3' end of the dsDNA in the presence of Mg2+ and Mn2+. Removes nucleotides from blunt ends, recessed ends, and nicks, but NOT overhangs!

Exonuclease VII

Degrades ssDNA from either the 5' or 3' ends
One of the few enzymes with 5' exonuclease activity.

Deoxyribonuclease I

From bovine pancrease, digests ss and dsDNA at pyrimidines. Typically used to remove DNA from RNA preparations.

Exonuclease II

Proofreading function of the pol
Degrades ssDNA from 3'-->5'

Southern Blotting

DNA is isolated and cut with REs. This allows investigators to determine the molecular weight of a restriction fragment and to measure relative amount in different samples.

Southern Blotting Procedure

1. RE digest DNA
2. Gel electrophoresis
3. Soak in HCl (depurinates, weakens H-bonds)
4. Soak in NaOH (denatures)
5. DNA transferred to a membrane
6. Immobilize (UV or bake)
7. Pre hyb to block
8. Hyb with probe

SyBr green

Non-specific intercalation into the minor groove of dsDNA, can be used in qPCR

Chaotropic agents

disrupts the structure and denatures the DNA by increasing the entropy and non-covalent forces like hydrogen bonds (ex. chemicals like - sodium iodide, or sodium perchlorate)

FRET probes

Fluorescence Resonance energy transfer - Distance dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

Molecular beacons

Measures accumulation of product at the annealing step
Contains target specific seq and inverted repeat that forms stem-loop
At annealing step probe hyb's to target, separating R and Q

Scorpion probes

PCR prod is covalently bound to dye; primer is bound to molc beacon type seq
After extension, target specific seq will unfold w/ the newly synthesized target seq, separating R and Q

Components of PCR

DNA template
Two primers that are complementary to the 3'
Taq polymerase
dNTPs
Buffer solution
Mg2+ (divalent cations) - higher Mn2+ concentration can increase the error rate during DNA synthesis

Thermal Cycling steps in conventional PCR

1. Initialization (94-96 C)
2. Denaturation (94-98 C)
3. Annealing (50-65 C)
4. Extension (70-80 C)
5. Repeat 2-4 ~30x
6. Final Elongation (70-74 C)
6. Final hold

Optimization of PCR methods

1. Check the Tm
2. Mg2+ concentration - too little can result in no PCR product, and too much may produce noise
3. PCR cycles
4. Add, extend, or increase the temp of the initial template denaturation step
5. Concentrations of other buffer components
6. GC Content 7. Taq concentration

Real-Time PCR/qPCR

PCR based method which is used to amplify and quantify a targeted DNA molecule. Enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when noramalized to the DNA input.