PCR Exam_HW

nucleotide composition

phosphate-sugar-base

nucleoside

base and sugar

dNTPs include which 4 things

dATP, dCTP, dGTP, dTTP

purines

A and G

“all gold”

pyrimidines

C and T

non-coding regions

introns

where are STRs seen?

introns

when looking at STR regions, how to design primers?

primers attach on 3’ and build new DNA 5’→3’

when you look at forsenics, what do you focus on?

“who can we not exclude?”

master mix includes what?

nucleotides (limiting reagent), reaction buffer, DNA polymerase (Taq polymerase)

cofactor for Taq

MgCl2

buffer purpose

makes pH stable, keeps DNA negatively charged, conducts electricity

purpose of Taq polymerase

attach to primer, build dNTP, can’t do its job if annealing doesn’t work

where do primers attach?

before STR area of interest

Taq is always at which temp

72°C

which cycles do you actually amplify things?

not until the 3rd cycle. cycles 1 and 2 are when you cut off the extraneous portions

how much DNA is made during PCR? (equation)

2^n

n= number of cycles

what causes DNA to move?

current, DNA charge, DNA size, gel pore size, buffer pH

what causes resolution?

size and charge of DNA

what enzyme causes the DNA structure to unwind and break down?

helicase

the more loci you look at, the greater ____ of ______ you get

power of discrimination

CODIS system

combined DNA Index System, a federally mandated database

looks at the same 13 STRs

technique used to detect DNA with a specific base sequence with a labeled hybridization probe to DNA bands immobilized onto nitrocellulose paper

southern blot

which process is this: hybridization performed on intact chromosomes

FISH (fluorescent in situ hybridization)

rtPCR is more ___ than standard PCR

sensitive

what can cause a PCR assay with no bands on any samples?

too few PCR cycles, malfunctioning PCR temp, stringency too high

purified DNA is indefinitely stable at what temp

-70°C

SNP with misplaced termination codon aka

nonsense variant

what does this describe: a variation in DNA in 1-2% of the population and may/may not affect protein function A

DNA polymorphism

composition of primer in PCR

oligonucleotide complementary to bases downstream of the target

how can a false neg PCR test caused by the presence of an inhibitor of the reaction be detected?

use internal control (amplification control)

what to do when: a PCR analysis of a vaginal sample gives neg result

amp control below cutoff

pos control above cutoff

neg control below cutoff

don’t report results!! repeat sample.

name of PCR product

amplicon

what parameter is used for genotype cell decisions?

cycle number at midpoint of curve

in rtPCR, which value is used to determine target number in specimen?

cycle number where the organism specific fluorescence begins logarithmic rise

in genetic analysis of cancer cells, why is loss of heterozygosity important

homozygosity for the same allele size causes mutation

if stringency is too low, what’s the solution?

increase annealing temp, che ck primer length and specificity, decrease MgCl2 concentration in master mix

primer dimers caused by

complementary primers to each other

adding DNA template to master mix and allowing to stabilize

primer dimers prevented by

keep reagents and PCR plate on ice bath

use hot start on thermocycler

PCR test of neg result in sample, pos control, neg control, and amp control?

neg, pos, neg, pos

after DNA performance with large 1200-1500bp, the bands are close to cathode. how to improve?

decrease % agarose concentration

if 35% of nucleotides are A, predict G

15%

why is it important to perform PCR on DNA from a crime scene?

small amounts of DNA get amplified for analysis

why do forensic labs analyze non-coding DNA and not genes?

DNA is “anonymous” and is <0.5% of human DNA that differs among people. they don’t control any known functions

master mix includes

oligonucleotide primers, 4 deoxynucleotides, reaction buffer (pH, salts, MgCl2),

components of PCR

taq polymerase, template DNA, master mix

why use polymorphic sequences in DNA?

use STRs, they’re unique

PCR 3 steps and temps

denature: 94*C

anneal: 42*C

extension: 72°C

how is temp of each PCR step determined?

if there’s a higher GC ratio, temp is higher at 98°C for denaturation

where do primers attach?

at 3’ end

why does DNA move through agarose gel?

electric current, size, buffer-pH

what factors determine migration of bands?

DNA size, length of DNA

2 techniques for PCR experiments

PCR amplification and gel electrophoresis

what’s needed to visualize DNA?

cellophane sheets, loading dye (ethidium bromide), 100x fast blast, tracing paper or acetate film

STR number of nucleotides and repeats

3-4 nucleotides

2-10 repeatsS

VNTR number of nucleotides

16+

SNP

single base pair polymorphism