Suzie Chen 2: genomic library + sanger gel electrophoresis + PCR

if upon cleavage by Bam HI human DNA is able to ANEAL with ITSELF can the strand of DNA be replicated in bacterial cells?

NO because if the DNA is anealed (comes back together) it CANNOT BIND TO PLASMID DNA

ALSO HUMAN PURE HUMAN DNA CANNOT BE REPLICATED BY BACTERIAL CELLS (has to become apart of the bacterial DNA)

what is the ideal outcome of cleavage by BAM HI for recombinant DNA production

Human DNA and plasmid DNA of bacteria get cut by the same restriction enzyme (same bps) and come together

Recombinant DNA now able to be replicated in DNA

DO NOT BIND WITH THEMSELVES UPON CLEAVAGE ‘

what would happen if after cleavage by BAM HI, the plasmid DNA and human DNA undergo dephosphorylation by phosphatase?

will they be able to replicate in bacterial cells?

do phosphatases increase or decrease the number of empty vectors?

HUMAN DNA AND BACTERIAL VECTOR WONT BE ABLE TO COME TOGETHER THROUGH PHOSPHODIESTER BONDS

• will NOT replicate

• REDUCE the number of empty vectors (fewer vectors are now available to bind with human DNA bc/ they don’t have the phosphorous to bind to human sugar)

If plasmid DNA anneals to itself after cleavage by BAM HI is it still able to replicate in bacterial cells?

Does this reduce or increase the amount of empty vectors / background?

yes, it can replicate since it is the same bacterial plasma DNA fragment it was before

INCREASE the amount of empty vectors/background number (are able to be

what are empty vectors?

what reduces the number of empty vectors?

what increases the number of empty vectors?

Plasmid that has everything needed to combine with human DNA but does not

PHOSPHATASE reduces empty vectors bc/ now the plasmid doesn’t have phosphorous to bind to itself

what happens when HUMAN DNA that reacts with phosphatase upon cleavage with BAM HI is added to bacteria? Can recombinant DNA be formed?

Is this DNA able to undergo replication in bacterial cells?

Bacteria will transfer phosphate to human DNA and will still be able to create recombinant DNA and replicate in bacterial cells

after you insert a piece of foreign DNA into bacterial cells, how can you tell which bacteria have the recombinant DNA?

colony hybridization

is it easy for recombinant DNA to be replicated once inserted into bacteria?

How can this be combatted?

NO usually only a minority retain the plasmids through replication

antibiotic-resistant gene within plasmid DNA

do cells automatically have antibiotic resistance when placed into bacteria?

NO you have to grow the bacteria in a medium that has the antibiotic in it so only those that are resistant will survive

_________ _________in plasmids provide resistance to compounds in the growth media allowing rare population of bacteria (that have taken up the recombinant plasmids) to grow while PREVENTING GROWTH OF EMPTY VECTORS

selective markers

what are three examples of selective markers?

• ampicillin

• tetracycline

• kanamycin

what are antibiotics

bacteria or fungi which inhibits a step of protein synthesis of another bacteria

in general terms what is a genomic library?

COMPLETE DNA of an organism is digested with a restriction endonuclease

EVERY SINGLE DNA FRAGMENT ARE IN VECTORS

the process of subdividing genomic DNA into clonable fragments and inserting them into vectors is called

creating a genomic library

the specific clones that carries the DNA sequences of interest of the genomic library must be _______ ___________ and __________

identified isolated and characterized

Construction of a Genomic Library :

Human DNA is cleaved with ______ ______

creating ________ of ___________ —→

Plasmids are cleaved with the same ______ ____________ as the human DNA —>

DNA fragments inserted into plasmids by _________ _______________ —→

Introduction of plasmids into ____________ —>

Genomic Library containing ALL _____ __________ of human DNA

• restriction enzymes millions DNA fragments

• restriction enzyme

• DNA LIGASE

• restriction fragments

Once you have your recombinant DNA and have transferred it into bacteria cells in a medium containing antibiotics what is the next step?

COLONY HYBRIDIZATION

what is the point of DNA hybridization?

be able to identify whether or not your gene of interest is present within a colony of DNA

probe (with the complementary strand to the gene of interest) will hybridize with the gene of interest

once you spread your colony of bacteria containing recombinant DNA in a master agar plate with antibiotics

• what do you place on top of the agar?

• what will happen after you place this on top of the agar plate?

• what is the next step?

• nitrocellulose/mylon membrane

• the colony will replicate onto the membrane

• remove the membrane now with new colony and soak in solutions and HYBRIDIZE W PROBE

in hybridization what do you place on top of the agar plate with the colony of bacteria?

nitrocellulose/ nylon membrane

in hybridization, once the bacteria is transfered onto the nitrocellulose/ nylon membrane and you remove it from the agar plate, what must be done to it? transfer

it must be

1. denatured (so probe can bind to it)

2. neutralized

3. washed

4. dried/ fixed

once the bacteria on the nitrocellulose membrane has been denatured, neutralized, washed, dried, and fixed

what can now be done?

bacterial DNA can be hybridized with probes (complementary strand of recombinant DNA) !

washed

autoradiographed

PROBE WILL ALLOW YOU TO SEE WHICH BACTERIA HAS RECOMBINANT GENE SO YOU KNOW WHAT TO GROW IN A LIQUID CULTURE

once a probe identifies which proteins from the membrane HAVE the recombinant DNA in them, what is then done?

the bacteria marked with probes is then purified and placed in a LIQUID CULTURE TO GROW

nitrocellulose/nylon membrane or “disc” allows DNA to be ________________ using ______/_____

THEN a single-stranded ______ of complimentary _________ RNA or DNA is added, and the strands are allowed to -________

denatured acids/bases

probe RADIOACTIVE re-anneal

DNA Hybridization

• the presence of a specific _____has to be identified before it can be used for further analysis

• restriction enzymes are used to reduce the DNA to smaller fragments

• the DNA segments are separated by ___________ ________ ________________

• to identify the fragment that contains the DNA of interest, a specific _____ is used to hybridize the DNA fragments

• gene

• agarose gel electrophoresis

• PROBE

how can you tell which nucleotides make up your DNA?

Sanger Chain - Termination DNA Sequencing

the Sanger DNA sequencing method uses __________ ________ to terminate DNA synthesis yielding series of DNA fragments whose sizes can be separated by electrophoresis

dideoxy nucleotides

In Sanger Sequencing we know

which dideoxy nucleotides terminated each fragment and which ____ it attached to

so we know the last _____ of each fragment and we will get EVERY SINGLE FRAGMENT

base

in gel electrophoresis do we read the bases from top to bottom or bottom to top?

bottom to top

each row you go up there is one additional base pair that did not get to go all the way down due to its larger size

the biggest fragment is at the top, as it was unable to reach the bottom because of its large size

does DNA sequencing provide the exact DNA sequence?

NO provides COMPLIMENTARY SEQUENCE

since dideoxy nucleotides are being detected and they pair with the template (they are the opposite base pair of the template)

do dideoxynucleotides have any OH’s at all attached to their 5 carbon sugar?

why is this important?

NO they have Oxygen embedded in the ring, phosphate and complimentary base but no OH like deoxyribonucleotide

since it doesn’t have OH you can not attach anymore nucleotides onto it and it terminates with the base attached to it matching the template strand

what are the starting materials to complete Gel Electrophoresis?

• single-stranded DNA

• radioactively labeled PRIMER

• DNA polymerase

• dideoxy nucleotides

what are the 4 dideoxy nucleotides mixed in with the single-stranded DNA, primer, and DNA polymerase in gel electrophoresis?

dATP

dTTP

dGTP

dCTP

what direction does gel electrophoresis run? bottom up or top to bottom?

In what direction do you start to read the COMPLIMENTARY strand that is shown by gel electrophoresis ?

top to bottom (biggest stay near top)

5” to 3” (bottom to top)

what was the template strand?

C TT G AA CGC AT

reading simply from bottom to top will give you COMPLIMENTARY so must use opposite to get template

For the SANGER METHOD do you mix the single stranded DNA, DNA polymerase, radioactively labeled primer, and all 4 deoxynucleotides ALL TOGETHER?

NO

you make one batch of DNA template strand + primer + Polymerase and you divide it amongst FOUR microtubes

one microtube has ddATP ddCTP ddGTP ddTTP

what and how do you load onto electrophoresis gel

there are 4 columns for each dideoxy nucleotide mixture

each column has either ATCG didoxynucleotide + DNA polymerase + Radioactive primer + DNA template strand

although you can’t visualize the size of the fragments of DNA you can tell the last base pair and the size of each fragment based on how far up it is on or down it is on the gel.

what must you do to a gel electrophoresis to see your results

autoradiography

Sequence of Gel Electrophoresis can be read from _______ on _________ and original template sequence ________

bands

autoradiograph

deduced

AUTOMATED DNA sequences can be done using ____________ _______

dye-labeled segments are applied to a ________ gel and subjected to electrophoresis

fluresent tags

CAPPILARY

Does SANGERs sequencing method deal with PCR?

NO NOT THE SAME THING

Sanger =DNA sequencing

PCR + DNA replication

_______ amplifies a region of DNA between two predetermined siters

Oligonucleotides complimentary to these sites serve as ________ for synthesis of copies of the DNA between the sites

PCR

primers

Each cycle of PCR doubles/quadruples the number fo copies of the amplified DNA

DOUBLES

_______ Polymerase comes from a bacterium that lives in hot springs

why is this SPECIFIC polymerase used in DNA replication during PCR

Taq

since it arises from hot springs it is able to work in hot temperatures

there is no DNA helicase, but DNA is separated by HEAT cranked up to 94 degrees celsius, other DNA polymerases would denature

what are the starting materials of PCR?

• double stranded DNA

• DNA primers

• taq polymerase

SEQ Polymerase Chain Reaction:

1. double-stranded DNA is denatured at _____ degrees

2. Primers anneal to each strand of DNA at what temperature?

3. Taq polymerase synthesizes the DNA strand at what temperature?

• 94 degrees

• 5 degrees below melting temperature (t m)

• 72

what is the flanking sequence in PCR and where is it found?

the sequence that the Primer will bind to

there are two on each strand 4 total

they are complimentary to DNA primer

one primer binds to the flanking sequence at one end of one DNA strand while another primer binds to the flanking sequence on the other end

do the primers have to be the same for both DNA strands? why or why not?

NO BECAUSE the start of the dna is not always going to be the same as the end AND they are complimentary to one another so they wouldn’t se the same

CATAGA
GCATCT

for example CAT not the same as TCT

does the amount of dna primer you add to the PCR initially matter? what about the size of the primer?

YES if you want MULTIPLE cycles then you should use EXCESS primer that will last for multiple cycles

you cannot add primer once PCR has begun to run

if you have a short primer than you will amplify a shorter sequence NEVER WANT TO LENGTHEN

• paternity testing and determining family relationships

• forensics DNA analysis

• Amplicfication of rare DNA

• human genetic testing

• cloning

• RT-PCR and qPCR (reverse transcriptase)

• diagnostic tests for disease causing pathogens

• human remains identification

all of the following are made possible through

PCR