Suzie Chen 1: CLONING (RECOMBITANT DNA)

Molecular biology involvees the development of tool and methods to manipuate DNA ___ _______ and ____ _____________

in vitro and in vivo

__________ _________ allow DNA to be cut at the precise sites into pieces

restriction endonucleases (restriction enzymes)

Cloning ________ such as plasmids carry inserted foreign DNA fragments to produce more ___________

VECTORS

proteins

Molecular biology deals with how to

• make ______ from the beginning

• identify ______ and determine treatment approaches

• __________ treatment modalities

• drugs

• diseases

• personalized

what has to been used to treat and prevent diseases in humans for 100s of years?

PROTEINS

________ therapy has been used for the treatment of tetanus and diphtheria

serum

tetanus and diphtheria antiserum was obtained from immunized _______ and __________

what is antiserum?

rabbits and horses

antiserum is blood containing antibodies

______ _______ started a large scale purification of __________ from cows and pigs to treat diabetes

Eli Lilly insulin

how are we able to use pig and cow insulin for ourselves?

high degree of sequence conservation

different species share the same sequences of DNA for functional proteins

the advancement of biotechnology has made it possible to move AWAY from ________-derived proteins to proteins with ________amino acid sequences.

away from animal

towards human

__________ proteins are less likely to cause side-effects and to elicit immune responses

recombitant

• monoclonal antibodies

• endogenous or modified HORMONES

• growth factors

• antisense oligonucleotides

• vaccines

• DNA / RNA for gene therapy

• stem cell therapy

are all examples of …

biotechnologically derived drugs

therapeutic proteins in comparison to _____ ______ drugs are

• more expensive

• different in size

• have high failure rates

• more personalized (ex. hercaptin + cetumixab/pantitumumab what do they do?)

small molecule

• hercaptin (HER2+ breast cancer patients)

• cetumixab and pantitumumab (colorectal cancer inhibit EGFR)

what do cetuximab and panitumumab do?

successful treatment using these drugs depends on what TWO things

• inhibit EGFR in metastatic colorectal cancer

  1. presence of EGFR on tumor cells

  2. absence of ADDITIONAL mutations downstream EGFR signaling effector molecules

why wouldn’t the monoclonal antibodies, cetuximab and panitumumab, be effective if there are mutations that exist downstream of EGFR?

if mutations are downstream then they are not impacted by EGFR and cause excessive growth independent of EGFR

ex. if PI3K was mutated it would cause increased growth regardless of whether or not EGFR is blocked as its mutation would cause it to work independent of EGFR

PRODUCTION OF THERAPEUTIC PROTEIN:

STEP 1: target selection

• how can you tell which human DNA sequence you want to imbed into DNA?

STEP 2: isolate _______ from ______that express the gene of interest

STEP 3: ______________ ___________ reaction

STEP 4: mixture of __DNAs

STEP 5: ________ selected __DNAs using PCR

STEP 6: CLONING INTO suitable ______ _________ (cut vector and Human DNA using same restriction enzyme and use ligase to seal together)

STEP 7: you know have a ______ DNA molecule !

STEP 8: transform the bacteria (ex. e.coli) for ________ (specifically _________ for e.coli)

1. search the gene using DNA database

2. mRNA cells

3. reverse transcriptase

4. c

5. amplify c

6. expression vector

7. RECOMBINANT

8. ampicillin resistance

THERAPEUTIC PROTEIN QUALITY CHECK:

Once you have made your recombinant DNA the next steps are

1. _______ the bacteria with the human DNA by selecting for ____________

2. quality check: ________ and ____________

3. transfection of ________ cells

4. stable expression is obtained by selection for ______ resistance

5. select for cells with the highest ____________ level

6. ____scaling of cell culture

7. PURIFICATION of the ___________ protein

• transform resistance

• sequence and orientation

• G418

• expression

• recombinant

QUALITY CHECK AFTER PURIFICATION OF RECOMBINANT DNA:

1. process validation (removal of _______ DNA and proteins, _______ clearance, ect)
   

2. consistency of the ________ process

3. consistency of the drug substnance (___________ + ________)

THEN, MOVE ON TO FORMULATION FOR HUMANS TO USE !

1. host viral

2. manufacturing

3. glycosylation + folding

MASS OF B- Globin IN HUMAN :

• how many base pairs are in each b-globin gene?

• how many daltons is ONE base pair (1/12 mass of carbon)

• so how many daltons is ONE b-globulin?

• how many beta globulin genes are in each cell?

• how many cells are there in the human body?

• what weight does b- globin hold in the human body?

• If there are 6.02 X 10²³ daltons per gram then how many grams are b-globin?
  

• 2000 bp

• 635

• 2000 × 635 = 1.27 × 10^6

• 2 copies

• 10^13

• (1.27 × 10⁶ x 2 × 10¹³ ) =

  2.54 × 10¹⁹ daltons

2.54 × 10¹⁹ / 1.27 × 10⁶ = 0.000042 grams

= 42 mcgrams

how many micrograms of b-globin are in the ENTIRE human body?

how many micrograms of b-globin are in ONE LITER of ecoli ?

42 micrograms

527 micrograms !

MASS OF b-globin IN e.COLI

• how many base pares make up b-globin in e.coli?

• how much in daltons does each basepair weigh in bacteria?

• so how many daltons are in each b-globin gene?

• how many copies of b-globin gene are in each e.coli cell?

• how many e.coli cells are in one liter of ecoli?

• so how many daltons of b-globin are in 1L of e.coli?

• if one gram = 6.02 × 10²³ daltons then how many grams are in 1 L of e.coli

• how many micrograms?

• 2000

• 635

• 2000 × 635 = 1.27 × 10⁶

• 500! (human is 2)

• 5 × 10¹¹ (human is 1 × 10¹³ )

• 1.27 X 10⁶ × 500 × 5×10¹¹ =

• 3.175 × 10²⁰ daltons

• 3.175 × 10²⁰ / 6.02 × 10²³ = 0.000527 grams

527 micrograms of b-globin in one liter of e.coli

each chromosome has a SINGLE long molecule of _______, within which are the sequences of individual genes

DNA

phosphodiester bonds connect the ______ from one nucleotide to the _______ of an incoming nucleotide

are phosphodiester bonds covalent?

sugar (OH of deoxyribose)

phosphate (one of three phosphates from nucleotide)

YES

do endonucleases (restriction enzymes) cut phosphodiester bonds or hydrogen bonds between nitrogenous bases?

phosphodiester bonds

how do endonucleases cut phosphodiester bonds?

hydrolysis of internal bonds within the polynucleotide chain

performs hydrolysis from the END of the polynucleotide chain

exonuclease

performs hydrolysis of the terminal ester bond that links a phosphate (diphosphate or triphosphate) to a terminal nucleotide at either 5” or 3”

performs hydrolysis of internal bonds within a polynucleotide chain

endonuclease

what is the difference between exonuclease and phosphatase?

phosphatase removes phosphate wheras exonuclease removes ENTIRE NUCLEOTIDE at the END of a DNA strand

restriction enzymes (endonucleases) bind to, recognize, and digest DNA WITHIN specific __________

called

__________ ____________ or

____________ ___________________

sequences

recognition sequence or restriction sites

Recognition sequences such as Taq1 and Smal1 are read the same backwards and forwards

what are they considered to be?

palindromes

what would cause a NOT enzyme to not bind to its recognition sequence?

if the recognition site is methylated

do all recognition sequences have the same amount of bases?

NO

different bacteria have different restriction sites that are made up of different numbers of bases

Which endonucleases result in

• blunt end

• 5’ overhang / 3’ recess

• 3” overhang / 5’ recess

DNA fragmentation

• Sma 1

• Bam HI

• Kpn 1

endonucleases that cut precisely opposite sites in the two strands of DNA

give an example

BLUNT

sma1

endonucleases cut asymmetrically within the recognition site such that a short SINGLE-stranded segment extends from the TWO 5’ ends

give an example

5’ overhang / 3’ recess

Bam HI (3 yr olds at recess destructive BAM and would say HI randomly)

endonucleases that asymmetrically cut within the recognition site resulting in a SINGLE stranded overhang from the TWO 3’ ends

give an example

3’ overhang/ 5’ recess

Kpn 1 (preeya apu KP nahar 1 is a young spirit but old (5’ older than 3 goes to recess)

based on where the restriction enzyme cuts how can you tell if its going to be a 3’ overhang or 5’ overhang?

if the 5’ end is cut farther along the way it is 5’ recess / 3’ overhang

you would think opposite!

farther along you are in life wouldn’t think you would go to recess

what is the probability of flipping a head twice?

are these event independent or dependent on each other and why?

how does this relate to DNA sequences and the number of nucleotides?

½ (first time) x ½ (second time) = ¼

independent bc/ getting a heads the first time doesn’t lower/heighten your chances of getting a heads the second time

more nucleotides you have the higher occurrence probabilities of DNA sequences

are nucleotide arrangements independent events?

YES one G does not impact the chance of getting a C A or T next to it

to find the possibilities of nucleotides you can have do 1 divided by (1/4) to the however many nucleotides you have power

1/4 because there are 4 different nucleotide options

what is the recognition sequence of EcoR1?

what is its occurrence probability? how did you figure this out?

GAATTC (youve GyAATT to C this

6 nucleotides and ¼ idependent chance to get each so 1/ (1/4) to the 6th power =

1/ 4096

HOW FREQUENTLY DOES RESTRICTION ENZYME CUT bc/ there are MULTIPLE restriction sites?

how many base pairs/ nucleotides are in DNA isolated from human cells?

if you want to get 1 million DNA fragments which is 1 × 10⁶ then you can use a restriction enzyme with 6 _________ (________)

3 × 10⁹ total / x (cuts every x nucleotide) = 1 × 10⁶ (fragments)

Overall endonuclease ________ has 6 ________ and cuts the sequence every ___________ base pairs giving you _______ DNA fragments from a total of 3× 10⁹ base pairs

3 × 10⁹

base pairs

EcoR1

EcoR1

base pairs

4000

If you have a piece of DNA with 300 base pairs if you use a restriction enzyme with 4 base pair recognition sequences such as Taq1 it will cut every 250 bases

How many times can a 300 base pair DNA get cut using Taq1 ?

once or twice

it can cut after 250 base pairs leaving 50 on the other side

OR

it can cute 250 anywhere out in the middle using two endonucleases leaving for example 5 bp then 250 bp then 45 bp = 300

DNA that can ACCEPT, CARRY, and REPLICATE other pieces of DNA when cloning

what is an example?

vector

example = plasmid DNA

are plasmids found in humans or bacteria?

what is their function and how do they do it?

BACTERIA

allow for antibiotic RESISTANCE so bacteria can survive

they encode for proteins/enzymes that inactivate antibiotics

this gene sequence in bacterial DNA allows for bacteria to stay alive by encoding for proteins that inactivate antibiotics

plasmid

the key to cloning a DNA fragment of interest is to link it to a _____/______ DNA molecule that can replicate within a ______ _________

vector/plasmid

host cell

vector/plasmid + DNA fragment of interest = _________________

which will then undergo ________ in ___________ ________ (human or bacteria?)

then we must ______, _________, and _________ the purified DNA fragment

RECOMBINANT DNA

replication in HOST cells (bacteria)

isolate, sequence, manipulate

EXAMPLE OF CLONING PROCESS:

EcoRI which is an_________ cuts both ________ DNA and ________ plasmid/vector

two fragments are joined by ________

e.coli cell is now transformed with recombinant plasmid. what does that mean?

the transformed cells are then plated onto a medium containing a ____________

only the _______ ________ plasmids are amplified and encode proteins to be used by humans

• endonuclease human bacterial

• ligase

• e.coli gained a new function with the human DNA

• antibiotic (ex. tetracycline)

• antibiotic RESISTANT (resistant colony)

CLONING SEQUENCED:

1. restriction enzyme cuts double stranded HUMAN DNA at ________ _______

2. these cuts produce DNA fragments with ________ _________ _______ ends (ready to mingle and bind with bacteria STICKY)

3. when two such fragements (human and bacterial DNA) are cut by the SAME restriction enzyme they can…

4. the joined fragements will usually form either a ___________ or ____________ molecule as well as other combinations

5. the enzyme ____ _________ is used to unite the _______ of the two DNA fragments producing a molecule of recombinant DNA containing human and plasmid DNA

1. restriction site / recognition sequence

2. hydrogen bonding

3. join through base pairing

4. circular OR linear

5. DNA ligase BACKBONE