PCR and nucleic acid extraction

lysis buffer

DNA extraction

-breaks apart cells

-inc 10 min at 55C

salt, SDS, EDTA, proteinase K

components of lysis buffer for DNA extraction

SDS

detergent in lysis buffer

EDTA

pH buffer in lysis buffer

proteinase K

enzyme in lysis buffer that dissolves proteins

100% EtOH

added to precipitate DNA as add to column

pos

silica is pos/neg charged

neg

DNA is pos/neg charged

salt, ETOH (56%)

components of first wash in DNA extraction

-break up H2O struct to make DNA less water sol

spin

what to do between each wash

EtOH (70%)

component of second wash in DNA extraction

-done at least 2 times

-spin between

-washes excess salt

Tris 

buffer used to elute purified DNA from column

-more pos charged than silica

EDTA

pH buffer protects purified DNA during elution

spectrophotometry

-measures quality and quantity of DNA from extraction

-nucleic acid abs: 260 nm

-protein abs: 280 nm

260

nm for nucleic acid abs in spectro

280

nm for protein acid abs in spectro

purity

measured by 260:280 ratio

1.8

optimal 260:280 ratio for DNA

2.0

optimal 260:280 ratio for RNA

50 ug/mL

amt of DNA present in one optical unit at 260 nm

40 ug/mL

amt RNA present in one optical unit at 260 nm

fluorometer

uses fluorescence to detect DNA concentration

-calibrated w each use

-inc light, inc DNA

4 mo

time of DNA storage at RT

1-3 yrs

time of DNA storage in fridge

7 yrs

time for DNA storage at -20 C

7+ yrs

time for DNA storage at -70

-20, -70

best temp to store RNA

1 mo

time for RNA storage in -20/-70C

DNA template, oligo primers, nucleotides, polymerase, buffers

elements of MMX for PCR

oligo primers

element of MMX that confer specificity for PCR

-20-30 bp

melting temp (Tm)

temp where ½ DNA is unwound

nucleotides

element of MMX

-equimolar of each

Taq

polymerase used for PCR
-heat stable

-do not vortex

buffers

component of MMX
-maintains optimal pH for enxyme activity

-MgCl2 and KCl, Tris, BSA

Tris (8-9.5)

pH buffer in MMX

BSA

buffer in MMX

-binds inhibitors to stabilize enzyme

DNA

DNA/RNA PCR requires thermocycling

denaturing (90-96)

first step of PCR

-opens up DNA

annealing (50-70)

second step of PCR

-primers onto target DNA

extension (65-75)

third step of PCR

-DNA synthesis/elongation by Taq

30

number of times DNA PCR is repeated

pos

PCR control that ensures enzyme is active, buffer is optimal, primers are binding right seq, thermal cycler works

neg

PCR control that ensures rxn is not contam

internal control (IC)

control that differentiates true neg from amplification failure

-inhibitor?

UNG amperase

PCR additive degrades RNA before cycling

multiplex

PCR that uses more than one primer paire

-panels: multigene, resp virus, stool pathogen, meningitis/encephalitis

-MRSA detection

MRSA

-multiplex PCR

-infectious org and antimicrobial resistance gene

-S. aureus and mecA gene

reverse transcriptase PCR (RT-PCR)

-RNA dep DNA pol

-RNA → cDNA

-RNA genomes (microorg), RNA expression, gene regions with large introns

real time PCR (qPCR)

-quantitative

-ethidium bromide or probes

-viral load, tumor load, treatment effects

-cycle threshold related to amt target seq

low

cycle threshold is low/high if amt starting target seq is high

high

cycle threshold is low/high if amt starting target seq is low

probe

detects presence of specific DNA fragment

-taqman, molecular beacon, scorpion primer, FRET
-fluorophore label

-hybridize close, but not overlapping with primer

-Tm higher than primers (want to stay during thermocycling)

-Ta lower than primers (want to anneal first)

Tm

probe Tm/Ta is higher than primer’s

Ta

probe Tm/Ta is lower than primer’s

taqman

-hydrolysis probe

-fluorescence with polymerase detachment

molecular beacon

-hairpin probe

-prevents fluor when close

-fluor before polymerase displacement (apart)

-viral loads: HIV, HepB, HepC

scorpion primer

-primer and hairpin probe

-fluor when sep

-BK/JC viruses (kidneys)

FRET

-proximity probe

-BCR-ABL t(9:22) in AML

-inc specificity in melt curve analysis → Tm detection

melt curve analysis

-final step of qPCR

-FRET for specificity

-mutations change Tm 

-factor V leiden, factor II prothrombin

FVL (exon 10)

gene mutation (where) in factor V leiden

G>A (1691, arg > gln)

SNP (mutation) in factor V leiden

factor V leiden

-thrombophilia → inc clotting

-FVL gene mut

-SNP exon 10

-1691 G > A

-Arg > Gln

factor II 

-second most inherited clotting abnormality

-F2 gene

-c.20210 G > A

-altered poly A tail → no stop → inc prot synth of prothrombin and clotting

-normal prot, abnormal amt

G > A (c.20210)

point mutation in factor II prothrombin clotting abnormality

F2

gene mutated in factor II prothrombin

txn mediated amplification (TMA)

-RNA-based

-isothermal

-billions RNA amplicons in <1 hr

-reverse txase and RNA pol

reverse transcriptase

enzyme used in TMA to convert RNA to dsDNA

RNA pol

enzyme used in TMA to convert dsDNA to cRNA

voltammetry

-target DNA

-ferrocene labeled signal molec

-test cardtridge with capture probe

-instrument measures signal from gold electrode to detect amt target molec in sample

-HepC treatment, CF, MTHFR (elevated homocysteine)

F508 del (3 bp del)

mutation that causes loss of CTFR function in CF

-detected using vollametry

MTHFR

enzyme that converts homocysteine to Met

-dec activity leads to atherosclerosis, thrombosis (heart attack risk), and dementia

-detected using vollametry

pyrosequencing

-short pieces of DNA, SNPs, point mut

-light upon addition of nucleotides

-add bp reagent one at a time and detect light

-hereditary hemochromatosis, EGFR, KRAS, BRAF

ATP sulfurylase

enzyme in pyrosequencing converts pyrophosphate released from bp addition into ATP

luciferase

enzyme in pyrosequencing reacts with ATP to produce light signal

apyrase

added to pyrosquencing wells to clean up unused nucleotides

hereditary hemochromatosis

-HFE gene, chromosome 6

-C282Y most common mutation

-H63D next

-C65S only signif if heterozygous with others

G > A (C282Y)

most common HFE gene mutation in hereditary hemochromatosis

C > G (H63D)

second most common HFE gene mutation in hereditary hemochromatosis

A > T (S65C)

rare and insignificant mutation on HFE gene in hereditary hemochromatosis unless hetero with H63D or C282Y

EGFR

-deletion in somatic cells leads to uncontrolled division

-exons 19, 20, 21 (codons 719, 768, 790, 858, E19 del)

KRAS

-codon mutations (12, 13, 61) lead to uncontrolled division and cancer

-leukemia, colorectal, pancreatic, lung cancer

BRAF

-exon 15 mut leads to uncontrolled cell growth

-V600E

-100% HC leukemia

-80% melanoma

-20-70% thyroid cancer

next gen seq

-human genome sequencing

-uses reference genome to detect common mutations

-more overlap = inc confidence