Module 11

Question 1

What did scientists wonder about bacteriophages in the 1950s? What was the solution?

Answer 1

why could they grow on some bacterial strains, but not others?

1960s: discovery of type I restriction endonucleases that recognize specific DNA sequences and cleave DNA

Question 2

Type I restriction endonucleases

Answer 2

• recognize specific DNA sequences and then cleave the DNA somewhere else
• restricts the entry of foreign DNA into bacterial cells

Question 3

Type II restriction endonucleases (restriction enzymes)

Answer 3

• first found in 1970
• cleave DNA within recognition site
• recognition site = palidrome
• much more useful

Question 4

What is a palindromic sequence?

Answer 4

a sequence of nucleotide bases that reads the same on the top strand as the sequence on the bottom strand of the DNA molecule in 5’->3’

Question 5

How is the nomenclature of REs derived?

Answer 5

from species (and strain) name and the order in which they were isolated

Question 6

Why don’t restriction endonucleases attack the host’s own DNA?

Answer 6

most common reason: host methylates a base in every copy of the RE site within its own site (REs do not recognize modified DNA)

Question 7

How are DNA sequences that are cut by Type II REs rejoined?

Answer 7

by DNA ligases; facilitated by H bonding between bases

Question 8

Cleavage patterns

Answer 8

REs can either cut directly in the centre of the recognition site, producing blunt fragment ends, or they can cut on the ends of the recognition sites, producing overhangs (sticky ends)

Question 9

What is gel electrophoresis used for?

Answer 9

sorting DNA (& RNA) fragments by size
- at neutral pH, DNA molecules are negatively charged because of phosphate groups
- in an electrical field, DNA will tend to move toward the positive electrode

Question 10

Why do we use a gel for electrophoresis?

Answer 10

because the gel prevents random diffusion of DNA

Question 11

Preparation of an agarose gel

Answer 11

1. prepare barriers in gel tray to retain the agarose
2. pour molten agarose into the tray
3. insert comb to form the wells before the agarose solidifies
4. load DNA samples in individual wells and apply voltage

*DNA usually contains dye to visualize

Question 12

EtBr

Answer 12

an intercalating dye that used to be used to visualize DNA in gel electrophoresis

*most DNA stains used now bind in minor groove

Question 13

Size-fractionation of DNA during agarose gel-electrophoresis

Answer 13

• shorter fragments migrate quicker
• migration of linear DNA molecules is inversely related to log of its molecular mass (or # of base-pairs)
• a standard curve of known size DNA fragments can be used to extrapolate the size (bp) of an unknown DNA fragment
• often 1st stage in characterization of an unknown DNA molecule

Question 14

What factors affect mobility of DNA fragments in gel?

Answer 14

1. agarose concentration in gel
2. topology (physical conformation) of DNA molecule
3. voltage
   - size

Question 15

How does agarose concentration affect the migration of DNA molecules?

Answer 15

• as it increases, pore size in gel matrix decreases
• smaller pores = more resistant to DNA movement, favour small DNA fragments, and give better resolution of size differences of small fragments

Question 16

What are the different topologies DNA molecules exist in, and how do these affect migration?

Answer 16

• linear: migrate as expected
• relaxed circular: migrate less than expected (appear bigger than expected)
• supercoiled (can be circular or linear; mostly negatively supercoiled): move more than expected (appear smaller than expected)

Question 17

Affect of voltage on migration through agarose gel

Answer 17

greater voltage speeds up migration rate of DNA fragments; too much voltage will heat up gel and cause depolymerization (bands looked smudged)

ex: ladder bands in lower voltage are scrunched up, more spread out in high voltage

Question 18

What are factors that do not influence the rate of migration of DNA molecules during agarose gel-electrophoresis?

Answer 18

%GC content or sequence of a DNA molecule

Question 19

How did the discovery of type II REs being able to cut DNA in predictable ways revolutionize molecular biology (2 ways)?

Answer 19

1. first easy way to study variation in DNA sequences, and map particular features
2. could easily recombine DNA sequences (with help of ligases) to create novel DNA sequences (birth of recombinant DNA technology)

*addition of agarose gel electrophoresis and EtBr staining also helped a lot

Question 20

What are the minimum requirements for DNA synthesis in vitro?

Answer 20

1. a template strand of DNA
2. a short, single strand of DNA complementary to part of the template (the ‘primer’)
3. DNA polymerase
4. deoxyribonucleoside triphosphates (dNTPs)
5. Mg+ (needed by polymerase)
   *DNA synthesis proceeds in the 3’ direction! (5’ => 3’)

Question 21

What was Mullis’ insight?

Answer 21

enzymatic copying of double stranded DNA using 2 primers, complementary to opposite strands could lead to exponential increase in amount of target sequence

Question 22

How many temperatures does PCR require DNA to be cycled through? How many cycles (generally)?

Answer 22

3; 30-35 to allow for a more than a billion-fold amplification of target DNA

Question 23

What 3 steps are in one cycle of PCR?

Answer 23

1. denaturation
2. annealing
3. elongation/ extension

Question 24

PCR: denaturation

Answer 24

temperature: 94-96
double stranded DNA denatures to single stranded DNA

Question 25

PCR: annealing

Answer 25

temperature: 50-65 (dependent on annealing/ denaturation temp of primers) - Tm is dependent on length and base composition of primers primers bind to complementary sequences

Question 26

PCR: elongation/ extension

Answer 26

temperature: 72 DNA polymerase (eg. Taq) binds to annealed primers and extends DNA at the 3' end of the chain

Question 27

PCR primers

Answer 27

- primers = short molecules of ssDNA (oligonucleotides/ oligos) usually 18-25 bases - priming between two oligos annealed to opposite strands can give exponential growth of product - size of PCR product depends on how far apart the annealing sites of 2 primers are - PCR products up to 40 kb have been produced but most involve 2 kb or less (yield drops with increasing length of DNA product)

Question 28

Why are primers usually 18-25 bases long?

Answer 28

- successful PCR depends on specific binding of primers to exact(!) positions that will allow amplification of target DNA - 'specificity' of primer binding is related to length of primer - shorter primers might not be specific enough in their binding - longer primers = more costly but offer an increase in specificity

Question 29

What are some applications of PCR?

Answer 29

- amplifying target sequences for further study => amplifying a target sequence within a complex mixture is like purifying the sequence (but need to know enough about sequence to design primers) - can detect rare sequences: as little as a single copy of DNA sequence, even in complex mixture - examples of rare DNA applications: detection of - bacterial contaminants in food, bacteria in environmental samples, pathogens or endosymbionts in organisms, forensics, environmental DNA (eDNA) - NOT good for detecting abundance of rare sequences (no idea what we started with)

Question 30

Stages of PCR (on graph)

Answer 30

- early cycles of PCR: production of DNA product is only limited by the amount in the previous cycle => exponential growth product - later cycles: dNTPs are less abundant, DNA poly starts to wear out, leading to slower growth of product => 'linear' phase - eventually: growth in amount of PCR product slows down greatly and then stops, as poly and dNTPs start to become exhausted => plateau phase - Cp/Cq marks first point product exceeds detection threshold of instrument (lag in collection of data, not in functioning of PCR)

Question 31

What phase on a log scale provides the best info on estimating the starting amount of DNA (or RNA) template?

Answer 31

log-linear phase

Question 32

qPCR

Answer 32

- growth in amount of PCR product is monitored by using a reporter dye, and a PCR machine capable of detecting fluorescence in each well - SYBR green used most, fluoresces more strongly when bound to dsDNA - binds primarily to minor groove in dsDNA

Question 33

Can you track multiple reactions in one reaction well using qPCR?

Answer 33

yes, by using a different colour for each target

Question 34

Applications of qPCR

Answer 34

- quantify amount of starting DNA of a particular sequence - measuring rate at which a particular gene is transcribed (need to convert mRNA to cDNA, use reverse transcriptase)

Question 35

The amount of PCR product (in ________ phsae of qPCR) is ________ to starting amount of DNA

Answer 35

exponential; proportional

Question 36

Reverse transcriptase process

Answer 36

1. oligo dT primer is bound to mRNA 2. reverse transcriptase (RT) copies first cDNA strand 3. RT digests and displaces mRNA and copies second strand of cDNA 4. double strand cDNA

Question 37

Why do PCR products end up being exactly the size bounded by the two primers?

Answer 37

after about 50 cycles, 75% of double strands are the same size

Question 38

What DNA sequencing remains the gold standard for accuracy and convenience today?

Answer 38

Sanger dideoxy chain terminating method

Question 39

What are the minimum requirements for DNA sequence in vitro?

Answer 39

- DNA primer - DNA polymerase - dNTPs - synthesis is in 5' => 3' direction

Question 40

What makes a dideoxyribonucleoside triphosphate different than a normal dNTP?

Answer 40

They terminate DNA synthesis because they lack the free 3' OH group (unable to extend chain further)

Question 41

How do we keep track of which bases are terminating at which fragments in Sanger Dideoxy?

Answer 41

attach different fluorescent colours to each type of ddNTP

Question 42

How do we sort the fragments produced by Sanger Dideoxy by size?

Answer 42

Use gel electrophoresis, the smallest fragments will represent DNA sequences terminating close to the primer

Question 43

Fluorescent dideoxy sequencing

Answer 43

- usually automated - gel electrophoresis uses denaturing polyacrylamide gel (contains urea) to separate ssDNA fragments by size - type of gel gives very fine resolution - as ddNTP-terminated fragments migrate in the gel, they pass a laser beam, that excites the fluorescent dyes, and a camera that records the flash of coloured light that results

Question 44

Sanger dideoxy sequencing pros and cons

Answer 44

pros: - very accurate - long sequencing reads - easy to do; can be automated - low cost (for small number of samples) - continues to be used for these reasons costs: - too slow for many application, like genome sequencing - costly when using lots of data - requires purification and preparation of each individual DNA sequence that is being studied - limitations led to invention of other methods, so called "next-gen" methods

Question 45

What was the main problem with Sanger dideoxy sequencing, and what was the solution?

Answer 45

problem: single sample sequencing is too slow solution: switch to massively parallel sequencing (next-gen sequencing, like illumina) - use variety of technologies, but all are sequencing by synthesis - allow for many of DNA segments to be sequenced at once (= massively parallel)

Question 46

What are the steps of illumina sequencing?

Answer 46

1. DNA fragmentation 2. Addition of primer binding and capture sequences 3. DNA denaturation 4. DNA immobilization 5. DNA amplification 6. DNA sequencing

Question 47

Illumina process

Answer 47

1. fragment DNA physically or enzymatically 2. add adapters with primers on inner end and capture sequences on outer end 3. denature DNA to separate strands 4. add DNA to flow cell 5. capture sequences will bind to short DNA sequences complementary to capture sequences that are pre-bound to flow cell surface 6. DNA strand is copied onto immobilized primer (single PCR cycle) 7. sample strand is discarded so now left with immobilized strand 8. cluster formation (amplification) => several PCR cycles 9. other capture sequences on cell act as primers for bridge formation during annealing phase 10. copy bridge in extension phase and separate strands during denaturation phase 11. get 1000 copies of each sequence in a cluster => remove strands from cell surface so that only forward or reverse strands remain 12. begin sequencing process => add one ddNTP as a time, take image to determine base, convert ddNTP to dNTP and repeat until all is sequenced

Question 48

Key points of illumina sequencing

Answer 48

- DNA must be short fragments - adaptor sequences added by ligation to ends of DNA segments => add sites for attachment of DNA sequencing primers and enable attachment to oligonucleotides on flow cell surface - DNA segments randomly dispersed over surface of flow cell - 'bridge amplification' used to amplify single DNA molecules into clusters of identical molecules - sequencing done by addition of one ddNTP at a time, labeled with fluorescent molecule, reversible! - computer interprets data from ddNTP fluorescent signals - millions of DNA sequences determined at once (massively parallel sequencing)

Question 49

Nanopore sequencing (3rd gen)

Answer 49

- not sequencing by synthesis - single molecule at a time (no pre-amplification by PCR) - enzyme unwinds DNA; single strand is pulled by an electric current through pore in a membrane - each base produces characteristic disturbances in electric current => used to read the base as it travels through

Question 50

Pros and cons of nanopore sequencing

Answer 50

pros: - long reads - no amplification step - small, very portable DNA sequencer that connects to USB port - can be used in field to get rapid results - can detect methylated bases cons: - slightly less accurate - other long read, single DNA molecule sequencing technologies exist