Tools of Molecular Biology

Question 1

what are tools for cloning DNA

Answer 1

enzymes for DNA and RNA manipulation

vectors for DNA propagation and expression

Question 2

what are tools for molecular characterization of mnucleic acids and proteins

Answer 2

Electophoresis, blotting, and microarrays

DNA sequencing and polymerase chain reaction

Question 3

How are the tools of human genetics applied

Answer 3

RFLP,xxxxxx

Question 4

what is cDNA

Answer 4

copy DNA made from mRNS (a protein coder) using reverse transcription

Question 5

where does reverse transcriptase come from?

Answer 5

mouse mammary leukemia virus

Question 6

what do restriction enzymes recognize

Answer 6

palindromic DNA sequences (usually)

Question 7

where does EcoR1 cut?

Answer 7

• –G A A T T C—
• –C T T A A G—

cleavage leads to sticky ends

—G AATTC—

—CTTAA G—

Question 8

biotechnological applications of Cas9

Answer 8

used to engineer the genomes of many different organisms

can cut RNA

used for “designer” therapeutics to target antibiotic-resistant bacteria

Question 9

recombinant DNA

Answer 9

all about cutting and pasting using restriction enzymes and DNA ligase

Question 10

transformation

Answer 10

process by which cells uptake target DNA for insertion into genome

Question 11

which direction does gel electrophoresis work?

Answer 11

from cathode (-) to anode (+)
small things migrate faster than big things in the gel

Question 12

how is DNA visualized in a gel

Answer 12

intercalating dyes such as ethidium bromide infiltrates into the DNA double helix

Question 13

how is ethidium bromide visualized

Answer 13

UV light, enhanced visualization when bound to DNA

Question 14

what must be present in a gel electrophoresis

Answer 14

size marker molecules (in kB) for reference to unknowns

Question 15

what determines temperature at which hybridization occurs

Answer 15

Tm of oligonucleotids

Question 16

Tm equation

Answer 16

Tm=2degC(A+T) + 4degC(G+C)

count number of each base pair in double strand, predict Tm

Question 17

why 2A-T but 4G-C

Answer 17

three h-bonds in GC, only 2 in AT

Question 18

How does Southern Blot work

Answer 18

allow detection of specific DNA fragments from complex mixtures via hybridization

Question 19

steps of Southern Blot

Answer 19

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

Question 20

which blot for DNA

Answer 20

Southern Blot (eponymous name) (cDNA of gene X to visualize)
not terribly sensitive

Question 21

which blot for RNA

Answer 21

RNA (cDNA of gene X to visualize)

Question 22

which blot for proteins

Answer 22

proteins (antibody to protein X to visualize)

Question 23

Where do restriction enzymes act on RNA

Answer 23

restriction enzymes do NOT work on RNA, only DNA

Question 24

microarray analysis

Answer 24

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

Question 25

what is the common sequence on every mRNAS

Answer 25

poly-A tail

Question 26

Sanger sequencing key reagent

Answer 26

Dideoxynucleotides

Question 27

Sanger sequencing key components

Answer 27

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

Question 28

Polymerase Chain Reaction (PCR)

Answer 28

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)

Question 29

rate of PCR propagation

Answer 29

exponential (doubling)

Question 30

substrates in PCR amplification

Answer 30

DNA template, DNA primers, deoxyribonucleotides

Question 31

how many strands in a DNA primer

Answer 31

1 (single stranded)

Question 32

RFLP

Answer 32

Restriction Fragment Length Polymorphism

Question 33

Single Nucleotide Polymorphisms (SNPs)

Answer 33

bi-allelic; ~1/50-300 bp; used for association

Question 34

principle of allele-specific PCR

Answer 34

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

Question 35

Variable Number Tandem Repeat (VNTR)

Answer 35

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