BIOL 2030: Module 11

Type I Restriction Endonucleases

• discovered in 1960s

  • originally thought to be rare, but now found to be very common

  • not very useful in molecular biology

• recognizes specific DNA sequences, and then cleave the DNA sequences somewhere else

• cuts DNA at specific sequences to create sticky ends

  • “restrict” the entry of foreign DNA into the bacterial cells

Type II Restriction Endonucleases

• restriction enzymes

• cleave DNA within the recognition site

  • this property has made them incredibly useful in molecular biology

• DNA sequences cut by them can be rejoined with ligases

  • ends are either 5’, 3’ overhang, or blunt

Difference Between Type I and II RE

• Type I cuts far from its recognition site (hundreds of bases away) and is a large, multi-subunit enzyme with both cutting and methylating functions, requiring ATP and SAM,

• Type II cuts within or very near the recognition site, is simpler, usually just a cutting enzyme, and is the most used in molecular biology for predictable results

Palindromic Sequence

• seen in type II REs

• sequence of nucleotide bases reads the same on the top strand as the sequence of nucleotide bases reads on the bottom strand of the DNA molecule in the 5’ → 3’ direction

RE Enzymes

• EcoRI

• BamHI

• HindIII

EcoRI

• Escherichi coli

• strain RY13

• 1st endonuclease isolated

BamHI

• Bacillus amyloliquefaciens

• strain H

• 1st endonuclease

HindIII

• Haemophilus influenzae

• strain Rd

• 3rd endonuclease isolated

Why dont bacterial restriction endonucleases attack the host’s own DNA?

• the most common reason is that the host bacterial cell methylates a base in every copy of the RE site within its own genome

Gel Electrophoresis

• a method for sorting DNA and RNA sequence fragments by size

• at neutral pH, DNA molecules are - charged because of phosphate groups

• in an electrical field, DNA will tend to move towards the positive electrode

• cannot be done in a liquid, as it needs to make a gel

  • most common kind is made from uncharged polysaccharide agrose

  • gel contains a buffer that provides ions a current which they can flow, and to keep the pH slightly above neutral

• “printed” gel is stained with a DNA binding fluorescent dye, EtBr

Size Fractionation of DNA

• shorter DNA fragments migrate more rapidly through the gel-matrix than longer molecules

• migration rate of linear DNA molecule is inversely related to log of its molecular mass, or # of its base pairs

  • meaning, larger molecules travel less distance and visa versa

• a standard curve of known size DNA fragments can be used to extrapolate the size of an unknown DNA fragment

• often in the 1st stage in the characterization of an unknown DNA molecule

Factors That Affect Mobility of DNA Fragments in a Gel

1. molecular mass (bp) of a DNA molecule

2. agarose concentration in gel

3. topology of DNA molecule

4. voltage

Concentration of Agarose in a DNA Molecule

• as agarose concentration increases, pore size in gel matrix decreases

• smaller pores means more resistance to DNA movement, favouring small DNA fragments, and giving better resolution of size differences of small fragments

DNA Molecule Topologies

• DNA molecules can exist in different topologies:

  • linear

  • relaxed

    • travels the least

  • circular

    • travels the least

  • supercoiled

    • travels the furthest

• topology of DNA strand affects its rate of migration in gels

Supercoiled DNA

• can either be circular or linear, but the ends of the linear molecule must be restrained

• in cells, DNA is often negatively supercoiled

  • + supercoiled DNA can be produced in vitro

Gel Electrophoresis Voltage

• greater voltage speeds up migration rate of DNA fragments during agarose gel-electrophoresis

What is meant by ‘sticky ends’ produced by REs and how do they help when DNA fragments are rejoined using ligase to produce recombinant DNA

Minimum Requirements for DNA Synthesis in Vivo

• a strand of DNA to act a a template

• a short, single strand of DNA complementary to part of the template

• DNA polymerase

• dNTPs

• Mg+

PCR

• polymerase chain reaction

  • has been called the most important technique in molecular bio

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

• requires DNA to be cycled repeatedly through 3 temperatures

  • this allows (assuming reaction occurs with 100% efficiency) for more than a billion-fold amplification of target DNA

Denaturation

• temperature 94-96^o

• double stranded DNA denatures to single stranded DNA

Annealing

• temperatures 50-65^o C

• primers bind to their complementary sequences

• T_m is dependent on length and base composition of primers

Elongation/extension

• temperature 72^o C

• DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain

PCR Ingredients

• deoxyribonucleotide triphosphates (dNTP’s)

• Mg²⁺

• primers

• template DNA

• thermostable DNA polymerase (often Taq)

• a salt

• Tris (pH control)

• stabilizers

PCR Primers

• short molecule of single stranded DNA, most often 18-25 bp long

• priming between two oligos (single stranded DNA) annealed to opposite strands can give exponential growth of product

• size of PCR product depends on how far apart the annealing sites of the 2 primers are

Length of Primers

• successful PCR primers are usually 18-25 bp long, as PCR depends on specific binding of primers to the exact positions that will allow us to amplify our target DNA

• specificity of primer binding is related to primer length

• shorter primers may not be specific enough in their binding, they may match and bind to multiple positions in the genomic DNA, resulting in amplification of incorrect DNA sequences

• primers that are 18-25 bp long are long enough to match only the intended DNA target sequence

Applications of PCR

• amplifying target sequences for further study

• detection of rare DNA sequences

  • but not good for determining abundance of these rare sequences

Stages of PCR

• during early cycles of PCR, production of DNA product is only limited by the amount in the previous cycle

  • exponential phase

• in later cycles, dNTPs are less abundant, and our DNA polymerase may start to wear out, leading to slower growth of product

  • linear growth

• eventually, growth in amount of PCR product slows down greatly and then stops, as polymerase and dNTPs start to become exhausted

  • plateau phase

C_p value

• marks the first point product exceeds detection threshold of instrument in PCR

qPCR

• quantitative PCR

  • how DNA is quantified in each cycle

• growth in amount of PCR product is monitored by using a reporter dye, and a PCR machine capable of detecting fluorescence in each well

• amount of PCR product (in exponential phase of qPCR) is proportional to starting amount of DNA

SYBR Green

• simplest and cheapest reporter dye in qPCR

• SYBR fluoresces much more strongly when bound to double-stranded DNA

• binds primarily to minor groove in double stranded DNA

Applications of qPCR

• quantify amount of starting DNA of a particular sequence

• measuring rate at which a particular gene is transcribed

PCR Set Up

What are the 5 minimum requirements for DNA synthesis in vitro?

• DNA template

• Primer (short ssDNA with free 3′-OH)

• DNA polymerase

• dNTPs (dATP, dCTP, dGTP, dTTP)

• Mg²⁺ (essential cofactor for polymerase)

What are the three stages of a PCR cycle?

• Denaturation (94–96 °C) – DNA strands separate

• Annealing (50–65 °C) – primers bind

• Extension / Elongation (72 °C) – polymerase synthesizes DNA

What happens to the target DNA content after each PCR cycle?

The amount of target DNA doubles (during exponential phase)

How many PCR cycles do you need? How many DNA copies are there?

• need 30-35 cycles

• Ideally: 2ⁿ, where n = number of cycles

  • (e.g., 30 cycles ≈ 1 billion copies, assuming perfect efficiency)

How do you “measure” the final amount of DNA product?

• Regular PCR: end-point measurement (e.g., gel electrophoresis)

• qPCR: fluorescence measured during each cycle

What is Taq

• A thermostable DNA polymerase from Thermus aquaticus

• Survives repeated heating to ~95 °C without denaturing

What direction is the new DNA strand synthesized (3’→5’ OR 5’→3’)?

• 5’ → 3’

In a mixture of DNA ... what provides the specificity of the target
sequence that will be amplified?

Primers

They determine:

• Which DNA region is amplified

• Product size

• Specificity of binding

What is the “usual” size of DNA fragment that can be amplified by PCR?

• Most commonly ≤ 2 kb

• Can go up to ~40 kb, but efficiency drops

What is the “usual” primer length? Why not shorter/longer?

18-25 base pairs

• Too short: bind nonspecifically → wrong products

• Too long: expensive, little gain in specificity

An invasive species produces a novel toxic protein ... you have isolated
this protein using chromatography, but you still don’t know what it is.
You successfully isolate DNA from the invasive species ... can you amplify the gene for this toxic protein?

• No — not without DNA sequence information

• You must know enough of the DNA sequence to design primers

Is “regular” PCR good for determining the abundance of a particular DNA sequence in sample?

• No

• Because:

  • End-point PCR reaches a plateau

  • Final product ≠ starting amount

Draw a PCR amplification curve

Axes:

• x-axis: PCR cycles

• y-axis: DNA amount (or fluorescence)

Four phases:

1. Lag phase

2. Exponential phase

3. Linear phase

4. Plateau phase

What is the relationship between the “exponential” phase and the “log-linear” phase?

They are the same phase, just plotted differently

What are the differences between the “linear” phase, and the “log-linear”
phase?

• Log-linear: high precision, constant doubling

• Linear: reagents limiting, variable growth

Which phase allows us to quantify the starting amount of DNA?

Exponential phase (log-linear)

• snapshots via fluorescent dye

• able to use a machine, as Cp is not yet reached

How do we monitor the growth of the product from one cycle to another?

Fluorescent reporter dyes

• SYBR Green (binds dsDNA)

• Fluorescent probes (target-specific)

What are the two key features that distinguish qPCR from (regular) PCR?

• Measures DNA during each cycle

• Allows quantification of starting DNA

Sanger Dideoxy Chain Terminating Method

• one of the two methods of sequencing DNA

  • invented in the 1970s, and still used today

  • remains the cold standard for accuracy and conviencience for sequencing small numbers of samples

Dideoxyribonucleoside Triphosphates

• ddNTP

• terminate DNA synthesis

  • whereas normal dNTP extends the DNA strand

• consider a DNA synthesis reaciton where 5% of the dGTP is replaced with ddGTP. this would give us DNA daughter strands of varying lengths, the lengths of which are determined by where the G’s occur in the sequence

  • we could do the same thing for the other bases (ddATP, CTP, TTP) so that we would get a subset of DNA elongation products terminating with a ddNTP base at every position in the DNA sequence

  • how would we keep track of which bases are terminating which fragments?

  • how do we sort out the different fragments by size?

How do we keep track of which bases are terminated by which fragments (ddNTP)?

• Fluorescent dideoxy sequencing

• we attach different fluorescent colours to each type of ddNTP, then use gel electrophoresis to sort the fragments by size.

  • the smallest fragments represent DNA sequences terminating close to the primer

• as ddNTP-terminated fragments migrate in the gel, they pass a laser beam, that excites the fluorescent dyes and a camera records the flash of coloured light that results

Sanger Dideozy Sequencing PROS

• very accurate

• relatively long sequencing reads

• easy to do, and can be automatefd

• low cost

Sanger Dideozy Sequencing CONS

• too slow for may applications

  • ex. genome sequencing

• costly when scaled up to aqquire lots of data

• requires purification and preparation of each individual DNA sequence that is being studied

Human Genome Project

• first human genome sequencing cost nearly $3 billion

  • mainly because the Sanger dideoxy sequencing was too slow and costly

• now a human genome sequence costs less than $1000

  • this is due to the switch to massively parallel sequencing

Massively Parallel Sequencing

• genomes are big!

• handling and sequencing individual samples is too slow for genome sequencing

• an approach was needed that allowed for many of DNA segments to be sequenced at once, and sequencing by synthesis

  • illumina

  • ion torrent

Illumina DNA Sequencing

• DNA needs to be short segments

  • this is accomplished by shearing/use of short PCR productrs

• adaptor sequences are added

• DNA segments are sequenced to be randomly arrayed across the flow cell surface

• bridge amplification is used to amplify a single DNA molecule into clusters of identical DNA molecules

• sequencing occurs by the addition of fluorescently labeled nucleotide analogs, 1 base at a time.

  • these dNTP analogs are chain terminators (like Sanger) but are reversible (unlike Sanger)

• after chemical treatment of the newly added dNTP, the chain can continue to elongate

• after each dNTP is added, sequencer pauses and exposes flow cell to a laser, and takes a picture to record what base was incorporated in each cluster.

  • process continues for a few hundred cycles

• computer interprets the data to infer the DNA sequence within each DNA cluster on the flow cell

• millions of distinct DNA sequences determined simultaneously this way

  • massively parallel DNA sequencing

Illumina Adaptor Sequences

• added by ligation to the ends of DNA segments

• adaptors add sites for attachment of DNA sequencing primers, and enable attachment to the oligionucleotides on the surface of the flow cell

3rd Generation Sequencing

• faster, single molecule, longer reads

• Nanopore sequencing

Nanopore Sequencing

• not DNA sequencing by synthesis

• single molecule at a time, therefore no pre-amplification by PCR

• enzyme unwinds DNA, a single strand is pulled by an electrical current through a pore in a membrane

• each base produces a characteristic disturbance in electrical current, which can be used to read the base as it travels through the pore.

Nanopore PROS

• long reads up to 1000 kb

• no amplification step to boost amount of template DNA before sequencing

• small, highly portable DNA sequencer connects to USB port on a computer

• can be used in the field to get rapid results

• can detect methylated bases

Nanopore CONS

• slightly less accurate than other methods

Is Each Method Massively Parallel?

Sanger

• No

Illumina

• Yes

Nanopore

• Yes

Is Each Method Sequencing by Synthesis?

Sanger

• Yes

Illumina

• Yes

Nanopore

• No

Is Each Method Single Molecule

Sanger

• No

Illumina

• No

Nanopore

• Yes

Is Each Method Chain Terminator?

Sanger

• yes

• non reversible

Illumina

• yes

• reversible

Nanopore

• no

Is Each Method Accurate?

Sanger

• most accurate

Illumina

• middle

Nanopore

• least accurate

• still up to 98-99% accurate

Read Length of Each Method

Sanger

• 650-1000 bp

Illumina

• 75-600 bp

Nanopore

• 100 kb