Mol Bio Numericals

Major steps in MOLDIO

Extraction and Purification

Amplification

Detection and Characterization

A single chromosome is considered a __ DNA molecule

1

3 components of nucleotide

a. 5 carbon sugar (ribose/deoxyribose)

b. phosphate esterified at 5' position

c. nitrogenous base at 1' position

Difference between nucleoside and nucleotide

nucleoside does not have phosphate

Pyrimidine Vs Purine

# of rings:

DNA substances:

RNA substances:

"Py, Thy, Cy"

Pyrimidine

# of rings: 1 ring

DNA substances: Cytosine, Thymine

RNA substances: Cytosine, Uracil

Purine

# of rings: 2

DNA substances: Adenine, Guanine

RNA substances: Adenine, Guanine

Pairs and their hydrogen bonds

Adenine/Thymine or Uracil= 2 hydrogen bonds

Cytosine/Guanine = 3 hydrogen bonds

Unit of measurement

single strand:

double strand:

short strand:

single strand: base

double strand: base pairs

short strand: nucleotides

2 reasons why DNA is more stable than RNA

1. double stranded

2. absence of OH group in carbon 2

In natural DNA replication, there are how many leading and lagging strands?

2 each

5 major enzymes in DNA replication

DNA Polymerase I: removes primers and leave a gap

DNA Polymerase III: adds nucleotides in primers

Helicase: unwinds the template strand

Primase: Attach to a strand, reads it, and makes primers

Ligase: Binds the gap left by the primer removed by DNA polymerase I

DNA polymerase 2 was removed because

It was only found in prokaryotes

Primers are 10-20 nt that provide a __ end

3'

DNA polymerization direction

3'-5' of the template

5'-3' of the growing strand

4 possible samples in NA detection

Cells in suspension (best)

Tissue samples

Microbial cultures

Cell-free samples

6 release or lysis methods

"MASHED"

1. Mechanical (grinding/mincing)

2. Heating/Boiling (95-100C)

3. Strong Alkali

4. Sonication

5. Enzymatic/Protease

6. Detergents

5 purification methods

a. Centifugation

b. Organic Isolation

c. Inorganic Isolation

d. Solid-phase Isolation

3. Chelating Ion-Exchange Resin

Organic isolation step (Phenol/Chloroform Isoamyl Method)

1. Add reagent to the sample

2. Centrifuge

3.Inorganic phase / Interphase / Organic phase

4. Pipet and transfer inorganic phase

5. Add isoamyl alcohol

6. centrifuge and decant

7. DIlute with TRIS-buffer or nuclease-free water

3 phases in Organic isolation step

Inorganic (Has the nucleic acids)

Interphase

Organic (Has the proteins and lipids)

Unique about Inorganic isolation (Salting-out)

Uses high salt, low pH conditions

Most commonly used purification step

Solid phase isolation

Solid phase isolation steps

1. Pour sample in a silica matrix bead and spin

2. Chaotropic salts weakens DNA hydrogen bonds to water making it attached to the silica matrix

3. Discard flow through and wash to remove other debris in the DNA-silica mixture

3. Elute the DNA from the silica

4. Dilute again

____% Chelex suspension is used in Chelating Ion-Exhange Resin

5-10

Main difference between Chelex and Solid Phase

Chelex adheres to contaminants; Silica adheres to the DNA

2 methods of mitcochondrial DNA isolation

1. Organelle differential centrifugation (Harvest the mitochondria)

2. Separate mtDNA from total DNA

a. Density gradient

b. PCR

c. Agarose electrophoresis

Why is RNA processing stricter than DNA processing?

RNAse are ubiquitous

After release and purification, what are 3 most common way to determine DNA quantity and quality

Spectrophotometer

Fluorometer

Electrophoresis

NA absorbs wavelength at

260 nm

Formula for DNA/RNA concentration

Absorbance x conversion factor

Conversion factor (mg/L)

dsDNA:

ssDNA:

RNA:

dsDNA: 50

ssDNA: 33

RNA: 40

Ratio of Relative purity determination

A260/A280

260- nucleic acids

280- proteins

Ideal RPD ratio

DNA:

RNA:

DNA: 1.8-2.0

RNA: 2.0-2.3

if less than the ratio, protein is contaminating

if more than the ratio, contaminating DNA/RNA from previous testing was present

Salt ratio

Ideal:

A260/A230

Ideal: 2.0-2.3

2 main factors in DNA/RNA electrophoresis

Size

Structure

Relationship of pore size to agarose concentration

Inverse

most common agarose concentration

0.5-5.0%

Main electrophoresis for small and large DNA fragments

small: PAGE

large: PFGE

3 steps in Capillary electrophoresis

Denaturation

Staining

Electrophoresis

All samples in capillary electrophoresis are

single stranded

Main tube used in capillary electrophoresis

Fused silica

Buffer pH of electrophoresis chamber

7-9

3 buffers used in electrophoresis

Tris Borate EDTA

Tris Phospate EDTA

Tris Acetate EDTA

4 denaturing agents in buffers

Formamide- block hydrogen binding sites

Urea- prevent folding

Methymercuric hydroxide- block amino groups

DMSO and Glycoxal - RNA

3 loading dyes

bromphenol blue

xylene cyanol green

orange G

3 density gradients

Ficoll

sucrose

glycerol

starting voltage

5-10 volts per cm

5 detection methods

a. Ethidium bromide (orange) - carcinogen

b. SYBR stain- non carcinogen

c. GelGreen/GelRed- more sensitive than the 2

d. Transilluminators

e. Silver stain - nonfluorescent but most sensitive (but complex technique)

Restriction enzyme target

restriction site

Restriction sites are ___ bp long with _______sequence

4-8 ; palindromic

3 types of Restriction enzymes

Type 1: cuts 1000 bp away from the site

Type 2: direct cut

Type 3: cut 35 bps away from the site

3 major steps in restriction enzyme mapping

1. Enzyme digestion

2. Electrophoresis

3. Mapping

Other term for nucleic acid hybridization is

probing

2 nuclease resistant probes

peptide nucleic acid probe

locked nucleic acid probe- 2' oxygen methylen bridged to 4' carbon

2 labeling methods of probe

1. radioactive (32P)

2. non-radioactive

a. fluorophores

b. chemiluminescence

c. digoxigenin

d. biotin

3 hybridization formats

Solid support/ Blotting

In situ

In-soltuon

5 types of solid support hybridization

1. dot blot and slot blot

2. Sandwich hybridization

3. Southern blot

4. Northern blot

5. Array-based hybridization

difference between standard and reverse dot blot

standard: nucleic acid is immobilized (multiple samples, 1 probe)

reverse: probe is immobilized ( 1 sample, multiple probes)

Unique in sandwich hybridization

1 capture probe

1 reporter probe

(Higher specificty)`

4 steps of Southern Blot

1. Restriction enzyme digestion

2. Electrophoresis

3. Membrane Transfer

4. Hybridization

Main difference of Northern and SOuthern Blot

RNA (Northern) does not need restriction enzyme digestion. Enzyme digestion is only for double stranded DNA (Southern).

RULE: Probes only hybridize to single stranded nucleic acids

3 blotting methods

1. Capillary transfer (gel carrying the NA at the bottom)

2. Vacuum transfer (gel carrying the NA at the top)

3. Electrophoretic transfer

Array based hybridization is like

Reverse dot blot

3 methods of in-solution hybridization are

a. RNAse protection/S1 mapping (strands hybridized are protected from S1 nuclease/RNAse)

b. Hybrid capture (Sandwich Hybridization, but in solution)

c. Hybridization protection (chemiluminescent probes hybridized with strands will be protected from alkaline hydrolysis)

Stringency that can lead to false positive or negative

High stringency: False negative

Low stringency: False positive

Definition of melting temperature in DNA

temperature by which 50% of the DNA are double stranded

Ideal hybridization temperature is ______ lower than the melting point

3-6 C

Formula for melting temperatures

(4 x GC pairs) + (2 x AT pairs)

Maximum salt concentration of stringency that can lead to constant hybridization

1.2 M NaCl

Acidic pH result in _______

Alkaline pH result in _____________

Acidic pH result in depurination

Alkaline pH result in dissociation

3 general categories of DNA amplification

1. Target

2. Probe

3. Signal

3 major types of Target Amplification

1. PCR

2. Loop-Mediated Isothermal Amplification / Autocycling Strand displacement DNA synthesis (isothermal)

3. Transcription-based Amplification (isothermal)

Who and when was amplification developed

Kary Mullis in 1983

6 basic components of PCR

Template DNA

Primer

dNTPs

DNA polymerase

PCR buffer

Thermal cycler

Which determine the specificity of PCR

primers

Length of primers

12-22 oligonucleotides

Primers are DNA/RNA?

DNA

2 problems in primers

1. Mispriming - priming other than the recommended target site

2. Primer Dimer - homology of both primers led to primers annealing with each other

4 DNA polymerase

1. Taq/Thermus aquaticus

2. Pfu/Pyrococcus furiosus

3. Tth/Thermus thermophilus (reverse transcriptase)

4. Vent/Themococcus litoralis (proofreading)

Amount of starting dNTPs

0.1-0.5 mM

Components of PCR buffer and function

a. Salt

b. MgCl

c. pH buffer

d. Bovine albumin

e. Formamide

f. Chaotropic salt

a. Salt: provide monovalent ions

b. MgCl: Provide Mg for enzyme activity

c. pH buffer: 8.0-9.5

d. Bovine albumin- enzyme stability and inhibitor binding

e. Formamide- lower melting temp

f. Chaotropic salt- reduce secondary structure formation

PCR mastermix

Buffer

dNTPs

DNA polymerase

Steps of PCR

1. Denaturation (94-95C for 15-30 seconds)

2. Annealing (50-65C for 30 seconds to 2 minutes)

3. Extension (72C for 1 to 2 minutes)

Number of cycles

25-40

PCR amplicons can be stored at

-20C

PCR controls and function

Positive- ensure thermocycler and reagent works

Negative- contamination

Negative template- mispriming

Internal amplification- inhibitors

Function of UV and psoralens

Psoralens make nucleic acids visible for decontamination by UV

dUTP-UNG method

dUTP will be added to PCR mix that may contain PCR contaminants from previous runs

UNG will then be added to destroy PCR contaminants

PCR starts by denaturation (destroys UNG)

Uracil-N-glycosylase incubation with dUTP in PCR mix

50C for 2-15 minutes

Hot-Start PCR methods

Wax Barrier

Heat-Activated DNA polymerase/primer/dNTPs

Goal is to prevent mispriming by separating template and enzyme (Wax barrier) or sequestering enzyme (Heat-activated)

Touchdown PCR is done by starting with annealing temperature ______ than the optimal binding temperature

higher (then decreased 1C for every cycle)

PCR product cleanup 3 methods

Shrimp alkaline phosphatase/ exonuclease I

Spin columns

Gel cutting and elution

PCR modifications

Multiplex PCR- multiple targets and primers

Reverse transcriptase- PCR

Nested/Seminested PCR

Realtime PCR/qPCR (thru threshold cycle)

3 primers for reverse transcription

1. Target specific primers (most specific)

2. Oligo dT primers (polyT primes polyA tails of mRNA; moderate specific)

3. Random hexamers (6-10 base ; least specific)

One start PCR uses ____ polymerase

Tth

How many primers are used in nested PCR

How many primers are used in semi-nested PCR

4

3 (1 of the first round primers was reused)

Refers to how many cycles it took for the levels to reach a threshold value

Threshold cycle

Relationship of the starting material from threshold cycle

Inverse

4 probes in real-time PCR

1. DNA TaqMan Probe (fluorescence during extension ; Taq polymerase separates reporter probe from quencher through its exonuclease)

2. Molecular beacon (fluorescence during annealing; separates reporter probe from quencher but is exonuclease-resistant)

3. Scorpion-type (fluorescence during annealing)

4. Fluorescence Resonance Energy Transfer/FRET (fluorophore + catalyst)

Steps in Transcription Based Amplification

RNA is encoded to dsDNA (by reverse transcriptase with RNAse activity)

dsDNA will be used to encoded RNA products (T7 RNA polymerase)

Components of TBA

RT with RNAse activity

primers with T7 promoter sequence

T7 RNA polymerase

rNTPs

dNTPs

RNA template (sample)

2 requirements for T7 RNA polymerase to work

dsDNA

T7 promoter sequence of primer