Tools of Molecular Biology

Question 1

We want to express recombinant human insulin in E. Coli… what would be our first step?

Answer 1

Take the insulin mRNA and reverse transcribe it to make cDNA

Question 2

Once we reverse transcribe insulin to give us c-DNA then what would we do

Answer 2

Then we need to polymerise the sense strand of DNA to make dsDNA

Question 3

One we have made insulin DNA then what do we do?

Answer 3

We use restriction digestion to cut at restriction enzyme sites

Question 4

In addition to using restriction enzymes on our insulin DNA, what else will we use the restriction enzyme on?

Answer 4

The expression vector (plasmid)

Question 5

In order for the insulin DNA to be incorporated into the complementary cuts on the plasmid we need one last enzyme to seal the deal…

Answer 5

Ligase

Question 6

What is it called when you put the recombinant DNA into the bacterial cell?

Answer 6

Transformation

Question 7

What does reverse transcriptase do?

Answer 7

Copies rna to dna

Question 8

In addition to the mRNA of insulin, what other substrates does reverse transcriptase require to make cDNA?

Answer 8

PRIMER (often oligo d-T primer)

d-NTPs

Question 9

Why are many primers oligo d-T?

Answer 9

Because easy to start at 3’ poly A tail

Question 10

What do restriction enzymes do?

Answer 10

Recognize and cut palindromic DNA sequences (usually)

Question 11

What is CRISPR

Answer 11

Is is an interference complex with RNA-guided DNA endonuclease (Cas9)

Question 12

What is the utility of Crispr Cas9?

Answer 12

It can use an RNA guide to cause targeted double strand breaks in the dna

Question 13

Key features of cloning vectors (3)

Answer 13

restriction sites
origin of replication 
selectable marker (e.g. amp)

Question 14

Plasmid as vector

general features

Answer 14

20kb

simple to clone, but transformation is inefficient

Question 15

Bacteriophage

general features

Answer 15

25kb

infection is 1000x more efficient

Question 16

Cosmid

Answer 16

45kb (Hybrid of phage and plasmid)

larger inserts

Question 17

BAC - bacterial artificial chromosome

Answer 17

300kb - used fro sequencing genomes and chromosome mapping

Question 18

YAC - yeast artificial chromosome

Answer 18

2Mb - used for sequencing of genomes and mapping

Question 19

Retrovirus

Answer 19

Used to deliver gene therapy

Question 20

How many ATP do we need to ligate our fragment into our plasmid

Answer 20

2 one for each strand

Question 21

Gel electrophoresis separates DNA based on?

Answer 21

Size

Question 22

How do we visualize DNA run on gel?

Answer 22

Intercalating dyes

e.g. ethidium bromide - only absorb UV light when within base pair

Question 23

Melting temperature - what does it determine

Answer 23

The temperature at which hybridization occurs

Question 24

Melting temperature (Tm) - definition -

Answer 24

The temperature at which 50% of oligonucleotide is hybridized to its perfect DNA compliment

Question 25

Tm = (equations)

Answer 25

2˚C(A+T) + 4˚C(G+C)

Question 26

Southern Blot

Answer 26

Allows detection of specific DNA fragments from complex mixture via hybridization

Question 27

In order to do a southern blot, after we run the gel what do we do?

Answer 27

denature the DNA duplex - then blot onto nitrocellulose membrane - then add excess (labeled) probe and reduce temperature to just below Tm to allow specific hybridization

Question 28

SDS-PAGE what is its function

Answer 28

gives protein an evenly distributed negative charge so that it will migrate toward anode based on size and not charge
Also breaks disulfide bonds

Question 29

Northern blot - detects?

Answer 29

Specific RNA sequences

Question 30

Western blot - detects?

Answer 30

Proteins via antibody targeting

Question 31

When would you use northern blot over pcr?

Answer 31

when you want to get the actual size of the fragment - which can be confusing when it comes to alternative splicing of the mRNA

Question 32

Pre-requisite for analysis of restriction digestion by gel electrophoresis?

Answer 32

All of the DNA you load in a well has the same sequence (ie multiple identical molecules)… unlike southern blot which can be used to detect specific DNA fragment in a complex mixture

Question 33

You calculate Tm based on …

Answer 33

Probe

Question 34

The probe for Northern blot …

is it sense or antisense?

Answer 34

antisense

because mRNA is sense

Question 35

The probe for Southern blot…

is it sense of antisense?

Answer 35

it doesnt matter because you have both strands of the DNA on the membrane

Question 36

Southern, Northern, and Western are useful for IDing what?

Answer 36

Whether there is a single specific gene, mRNA, or protein in a complex mixture, respectively

Question 37

What is microarray analysis

Answer 37

You anneal probe to glass slide … then put in DNA/RNA to interrogate.. that is labeled with a fluorophore… it will hybridize to the probe on the glass slide that it is complementary to and light up

Question 38

Why was microarray developed

Answer 38

To simultaneously analyze the expression pattern of thousands of genes

Question 39

Microarray chips contain hybridization probes that are complementary to…?

Answer 39

fluorescently labeled nucleic acids from source of interest

Question 40

Microarray analysis allows comparison in ….

Answer 40

global gene expression between different conditions (i.e. tumor vs normal)

Question 41

DNA sequency… key reagent?

Answer 41

dideoxynucleotides- because they will terminate the chain

Question 42

Do you need to know anything about your template to perform sanger sequencing?

Answer 42

Yes you need to know enough to make a primer

Question 43

Why would Sanger Sequencing be useful for heterozygotes at given base position?

Answer 43

Because we will get a mixed peak (50% blue and 50% red) whatever color - letting us know that they are heterozygote

Question 44

Why do PCR?

Answer 44

Most sensitive way to search for a sequence in a complex molecule

Question 45

Do you have to know anything to do PCR

Answer 45

Yes, again you need to make a probe to both sides

Question 46

After n rounds of PCR how many copies will you have?

Answer 46

2^n

Question 47

At what temperature do you start PCR

Answer 47

95 - this will denature the DNA to yield two strands

Question 48

Once you denature the DNA, at what temperature do you anneal?

Answer 48

55 ish - this will allow hybridization of probe

Question 49

Once you allow probe hybridization, at what temperature do you elongate?

Answer 49

72 - this is the optimal temperature for the bacterial polymerase

Question 50

Ebola is a (-) sense ssRNA virus. What molecular tools would you use to detect the virus in the patient’s serum, and in what order?

Answer 50

Reverse transcription –> PCR –> gel electrophoresis

Question 51

Can you do PCR on RNA?

Answer 51

Only after doing rever transcription - because the enzyme require DNA - it cannot copy RNA

Question 52

The substrates in PCR amplification are:

Answer 52

DNA template, DNA primers, deoxyribonucleotides

Question 53

RFLP can be used to diagnose diseases… How so?

Answer 53

The mutation of a restriction site (e.g. an A–>G mutation) can result in skipped fragmentation - thus upon running on gel, the fragment will not have been fragmented :)

Question 54

Example of RFLP diagnostic

Answer 54

Sickle CELL

Question 55

Diagnostic use of restriction enzymes for RFLP uses what kind of blot?

Answer 55

Southern

Question 56

Allele Specific PCR

Answer 56

Interrogates Genome via allele specific primers - the very 3’ position is the quarry position - depending on whether WT or Mutant - will hybridize with corresponding primer - only those hybridizing will undergo PCR
Obviously you will need to run two tubes - each with unique primer