Tools of Molecular Biology Flashcards
what are tools for cloning DNA
enzymes for DNA and RNA manipulation
vectors for DNA propagation and expression
what are tools for molecular characterization of mnucleic acids and proteins
Electophoresis, blotting, and microarrays
DNA sequencing and polymerase chain reaction
How are the tools of human genetics applied
RFLP,xxxxxx
what is cDNA
copy DNA made from mRNS (a protein coder) using reverse transcription
where does reverse transcriptase come from?
mouse mammary leukemia virus
what do restriction enzymes recognize
palindromic DNA sequences (usually)
where does EcoR1 cut?
- –G A A T T C—
- –C T T A A G—
cleavage leads to sticky ends
—G AATTC—
—CTTAA G—
biotechnological applications of Cas9
used to engineer the genomes of many different organisms
can cut RNA
used for “designer” therapeutics to target antibiotic-resistant bacteria
recombinant DNA
all about cutting and pasting using restriction enzymes and DNA ligase
transformation
process by which cells uptake target DNA for insertion into genome
which direction does gel electrophoresis work?
from cathode (-) to anode (+) small things migrate faster than big things in the gel
how is DNA visualized in a gel
intercalating dyes such as ethidium bromide infiltrates into the DNA double helix
how is ethidium bromide visualized
UV light, enhanced visualization when bound to DNA
what must be present in a gel electrophoresis
size marker molecules (in kB) for reference to unknowns
what determines temperature at which hybridization occurs
Tm of oligonucleotids
Tm equation
Tm=2degC(A+T) + 4degC(G+C)
count number of each base pair in double strand, predict Tm
why 2A-T but 4G-C
three h-bonds in GC, only 2 in AT
How does Southern Blot work
allow detection of specific DNA fragments from complex mixtures via hybridization
steps of Southern Blot
1) cleave DNA with restriction enzymes, separate on gel
2) Denature DNA duplex
3) capillary action to transfer DNA from gel to nitrocellulose
4) hybridize with labeled DNA or RNA probe (add excess probe and reduce temperature to just below Tm to allow specific hybridization
5) develop radiofilm to expose labelled probes
which blot for DNA
Southern Blot (eponymous name) (cDNA of gene X to visualize) not terribly sensitive
which blot for RNA
RNA (cDNA of gene X to visualize)
which blot for proteins
proteins (antibody to protein X to visualize)
Where do restriction enzymes act on RNA
restriction enzymes do NOT work on RNA, only DNA
microarray analysis
old technology
developed to simultaneously analyze the expression patterns of thousands of genes
microarray chips contain hybridization probes complementary to fluorescently-labeled nucleic acids from source of interest
comparisons in global gene expression can be made between different conditions
what is the common sequence on every mRNAS
poly-A tail
Sanger sequencing key reagent
Dideoxynucleotides
Sanger sequencing key components
5’ Primer of known sequence to hybridize to template DNA strand
4 base nucleotides
separate into four flasks, each with its own dideoxynucleotide (A,T,G, or C), which acts as a polymerase “poison”
create fragments of different lengths, end determined by poisoned nucleotide randomly
measure lengths of all available strands in each tube, shows you location of nucleotides
Polymerase Chain Reaction (PCR)
denature duplex DNA
First cycle - anneal (attach) primers (must have some pre-info to create good primers)
Use polymerases from thermophilic organisms to extend DNA copy at high temp (elongation) (taq is thermus aquaticus)
rate of PCR propagation
exponential (doubling)
substrates in PCR amplification
DNA template, DNA primers, deoxyribonucleotides
how many strands in a DNA primer
1 (single stranded)
RFLP
Restriction Fragment Length Polymorphism
Single Nucleotide Polymorphisms (SNPs)
bi-allelic; ~1/50-300 bp; used for association
principle of allele-specific PCR
measure whether specific mutation is at a specific site
have a common downstream primer, then both a wild-type and mutant-type primer
run two tests, one with each primer
primer mismatch means no PCR
heterozygous presentation means both primers will work in the sample, since one wild-type and one mutant-type allele are present
Variable Number Tandem Repeat (VNTR)
take advantage of variable length non-coding DNA segments. Apply a common primer, then look at strand length. The different lengths of the non-coding strands can uniquely identify an individual, used for crime scene identification
