New Material Final Exam Lecture 4 & 5/5: DNA Analysis - Parts 1 and 2

• There are a lot of details to high-throughput DNA sequencing that we
  did not cover in class. You are only responsible for the level of detail
  we covered in class.

• You should know the components of a plasmid used in transformation
  experiments and why each is used.

• You should know how blue-white screening works.

• You should know the components of a PCR and the steps in each cycle

what is the diff between cloning and molecular cloning

cloning → cloning entire individual

molecular cloning → using some sort of vector (eg plasmid) then putting it in a cell that can naturally make copies of it

• recombinant plasmid is inserted into microbe

• microbe reproduces, reproducing plasmid

what are endonucleases

cut phosphodiester bonds WITHIN nucleic acids (not on ends)

what are restriction enzymes / restriction endonucleases

subtype of endonuclease

• named for enzyme’s natural role in restricting infection by phages

• recognizes foreign DNA and cuts it into pieces

• restricts what type of DNA can exist in the host

what are palindromic strands of dna

sequence reads the same in the 5′ → 3′ direction on both strands

what do type 2 REs (restriction enzymes) do

Recognize a specific short DNA sequence (usually 4–8 base pairs, often palindromic).

• Cut both DNA strands within or very close to that recognition site.

• Produce either:

  • Blunt ends (straight cut)

  • Sticky/cohesive ends (staggered cut with overhangs)

• each RE has a unique recognition sequence

what are blunt and sticky ends respectively

enzymes that prod blunt ends: cut straight through both strands at the same position, leaving no overhangs

enzymes that prod sticky ends: cut offset between the two strands, creating short single-stranded overhangs

whicj direction is the recognition site for restriction enzymes always read in

5’-3’

what does gel electrophoresis do? how does it work

separates dna fragments based on size

• have aggross gel w wells filled w dna-binding blue dye in it

• the matrix is mostly made of aggros

• has + and - electrodes

• during electrophoresis, blue dye moves down the gel

• the dna moves down too (diff distances depending on size)

  • DNA migrates from - charged end to + (bc dna is - charged)

  • the larger the dna molecule, the slower it moves through the gel matrix

• DNA-binding dye lights up DNA bands under UV light

do larger or smaller molecules move further towards the + side of the

smaller

what does each band represent in gel electrophoresis

many dna fragments of the same size

what is molecular cloning

the process of using living cells to make many exact replicas of a DNA fragment

how are dna fragments purified and inserted into vectors (eg plasmids)

1. purify a DNA fragment (eg by gel electrophoresis)

2. insert the dna fragment into the vector

• digest a pre-existing vector (eg plasmid) AND the dna fragment with the same RE (opens them up, leaves them w same exposed complimentary regions of dna)

  • creates complimentary sticky ends that can H+ bond w each other

• DNA ligase joins the dna fragments tg (covalently links them)

• cell divides and reprod plasmid with gene of interest interested in it

explain the steps to molecular cloning

1. purify dna fragment (eg by gel electrophoresis) and insert it into a vector (eg plasmid)

2. transfer vector w its insert into “compitent” cells (cells that can incorporate foreign substances into it and replicate foreign dna)

   • copies will be made through dna replication

will inserting a segment of dna into a plasmid yield identical new plasmids

no → you will end up w a combination of diff plasmids

• some proportion of plasmid will have insert in one orientation or the other

• some will have no insert (not 100% effective)

what are the 3 main features of plasmids used as cloning vectors

1. origin of replication

2. selectable marker gene (eg antibiotic resistance gene)

   • some way to differentiate btwn cells that uptook plasmid vs ones that didn’t

     • if they took in the antibiotic then you can treat the cells created w the antibiotic and the surviving ones would have uptaken the dna

3. polylinker (DNA sequence containing multiple restriction enzyme recognition sites)

   • so could open up the plasmid

what is bacterial heat shock transformation

process of inserting the plasmid vector into the bacterial cells

• making bacterial cell walls leaks so they let in plasmids

  • make test tubes a bacterial cells of interest really cold then really hot suddenly → damages cell walls

what is done to the bacterial cells of interest after heat shock transformation

• shake them to whatever temp the bacteria grows best in in their test tube with NO ANTIBIOTIC IN THE INITIAL GROWTH MEDIUM

  • gives cells an hourish to develop / incorporate resistance genes from plasmid

• add them to a growth medium +antibiotic

  • so only cells that have properly incorporated the plasmid survive

  • creates colonies instead of a lawn

what are inserts

the piece of foreign DNA you WANT to put into the plasmid vector

• NOT the antibiotic resistance gene → that is added in conjunction to that

what are some challenges with creating molecular clones

1. not all cells will take up a plasmid

2. not all plasmids will have an insert (self-ligation)

   • some cells will ligate before plasmid can be incorperated

how do you distinguish between cells without inserts from those w inserts

the lacZ gene (codes for B-galactosidase; breaks down lactose)

• on some plasmids the multiple cloning site is located within the LacZ gene

• lacZ gene is split by fireign DNA insert

• no LacZ product (no B-galactosidase)

  • B-galactosidase can also cleave things other than lactose → can cleave X-Gal (analogue of lactose) and make it from white → blue

• put vectors onto plate with X-Gal → ones with LacZ gene in tact will show as blue and ones w no LacZ (and WITH insert) will be white

how do you distinguish between cells…

• without plasmids

• without inserts

• with plasmid with insert

• without plasmids → antibodies

• without inserts → blue

• with plasmid with insert → white

what are some applications of molecular cloning

• gene expression

  • prod a lot of protein product of interest

  • study gene fx

• introduce and study specific muts

• link promoter regions to reporter genes to study gene regulation

• etc

what is a polymerase chain reaction (PCR)

allows us to amplify regions of DNA we’re interested in

• in vitro (tube) DNA synthesis reaction

• creates many copies of a segment of dna

  • generally used to amplify segments of dna ~200-1000 bps long

• needs very few starting dna molecules

  • very sensitive

• can be visualized using agarose gel electrophoresis

what are amplicons

amplified dna regions in PCRs

what are some applications of PCRs

make many copies of dna sequence of interest

• research

• genetic engineering

• cloning experiments

used in research and diagnostics

• prenatal genetic testing

• testing food for pathogens

• HIV test

genetic fingerprinting

etc

which reagents are needed in PCRs

• polymerase

• do NOT want primase (will create primers on random pieces of dna)

  • instead want to provide specific primers in the mixture

• DNTPs needed

• need template

• need buffer

what are the 3 steps in each cycle of polymerase chain reactions (PCRs)

1. denaturation

   • seperate double strands using temperature

2. annealing

   • make conditions so primers can add

3. extension

   • synthesizing new dna

explain the denaturing step in PCR reactions

around 95-98 degrees C

• double stranded dna breaks apart

  • mixture contains prokaryotic dna pol from thermophiles so they don’t denature

explain the annealing step in PCR reactions

around 50-65 degrees C (depends on length and sequence of primer)

• primers now can/do bind to unwound single stranded dna

explain the extension step in PCR reactions

around 68-72 deg

• dna pol binds and extends off primer

what does PCR result in? why is that the case

exponential amplification

• dna doubles each time

  • longer you keep PCR going the more times there will be more replications

what is the general PCR thermal cycle

1. keep temp around 95 to 98 deg C at first for around 2 mins so dsDNA breaks apart

2. reduce temp (to around 50-65 C) for around 20 secs so primers can bind

3. inc temp again to around 68-72 deg C so polymerase can bind and extend off primer

repeat around 30-40 times

• dec temp to around 4 C when done

what was the first DNA sequencing method? explain what it is used for and what it is a combination of

Sagner sequencing

• older method

• used for smaller sequences (not whole genomes → although USED to be used for that but took ages)

• combines chain termination with florescent labeling

what is chain termination

• DNA pol connects the 3’ OH to the next nucleotide at the 5’ phosphate

• strand cannot elongate without the 3’ OH

  • cant add other nucs if that OH is turned into an H instead

what are dNTPs vs ddNTPs

dNTPs are standard → they have a OH group on the 3’ end

ddNTPs (deoxi nucleotide triphosphate) → has OH switched with an H can’t add another nucleotide after these

what is fluorescent labeling

• each ddNTP labeled w a diff colour

• dNTPs are NOT labeled

• the ID of the last base can be determined based on how the nucleotide fluoresces (which colour it is)

are dNTPs or ddNTPs more abundant in Sanger sequencing

dNTPs → higher conc so more likely to encorperate

how do you get so many diff dna sequences of diff lengths in Sanger sequencing to test from

PCR with fluorescent, chain terminating ddNTPs

what happens when a ddNTP is incorperated into a nucleotide sequence during dna repilcation? what does this help with

replication stops → nothing else can bind

• you end up w a mixture of strand lengths each ending where the ddNTP has been incorporated

• can tell which is where in the seq based off colour

  • if only dNTP, would all be same length and be harder to tell which nuc is where

t/f: incorporation of the ddNTP is random

true

what are 2 potential outputs of Sanger sequencing

1. sequence chromatogram

2. text file

   • computer’s interpretation

   • errors common near the start and end (bc peaks are closer tg → less chance ddNTPs bind there)

note: the pic is a sequence chromatogram

what is capillary electrophoresis similar to? what does it do?

similar to gel electrophoresis BUT not agarose and in a thin tube

• reaction products separated by size

• each fragment size has a fluorescent label

how does capillary electrophoresis work?

1. PCR w fluorescent, chain-terminating ddNTPs

2. size seperation like in capillary gel electrophoresis (but recognized

3. laser excitation and detection by sequencing machine (tells which colour each ends w → which nucs are where in the overall sequence)

which method would you use if you wanted to sequence more than one DNA segment at a time - like a whole genome or all the mRNA in a cell

high-throughput sequencing

• CAN use sanger but takes forever (have to break up whole genome into segments and separately sequence each)

what are the 2 main technologies used for high-throughput sequencing

1. Sequencing by synthesis (Illumina)

   • PCR-based dna sequencing on a flow cell

   • diff machines exist that have diff read lengths, cycles, number of samples that can be included, and amount of data you get out (sequencing depth)

2. (Oxford) Nanopore technology

   • based on characteristic current disruption each nuc produces when a dna molecule flows through a nanopore

   • diff machines exist → including the Minlon which is the size of a usb key

nanopore looks mostly like Sanger

what is a library and the library preparation process in sequencing by synthesis

library: the collection of DNA (or cDNA) fragments from your sample that have been processed so they’re ready to be sequenced

• contain adapters that help w the process and primers

a sequencing run can include many samples, each usually consisting of many DNA fragments w different sequences

• one index per sample NOT one index per type of fragment

• can have a mixture of 10s of hundreds of diff sample

what does sequencing by synthesis use

reversible dye terminator nucleotides

• have reversible blocking group → can be manipulated chemically to regenerate an -OH allowing synthesis to continue one step at a time

• a diff fluorescent dye for each nuc

like Sanger sequencing but reversible

t/f: each sequence in synthesis sequencing will all be the same length at any given time

true

what does the output of sequencing by synthesis look like

each dot is many identical copies of a single dna fragment from the library

• diff channels for each nuc w a bunco of dots on it

• tells you which place in the sequence it is

• each dot lights up according to the nucleotide just incorporated

• all samples are sequenced at the same time

explain nanopore sequencing

flow cell contains an array of tiny holes (nanopores) embedded in a mem

• each nanopore has an electrode connected to a sensor

• when a molecule passes through a nanopore, the electrical current is characteristically disrupted → as DNA moves through it will influence nanopore differently and computer connected to electrode will tell you which is nucleotide is which (diff electrodes connected to diff nanopores let off diff signals which tells which nuc it is)