DNA Technology

PCR: ingredients

target sequence, Taq polymerase, magnesium ions, dNTPs, primers, eppendorf tube, thermocycler

PCR: purpose/application

to amplify a sequence/ make many copies of a specific sequence of DNA. This is to solve the problem that there is usually not enough DNA of a specific sequence to use or show up as a band in gel electrophoresis.

PCR: process

eppendorf tube holds all DNA (containing target sequence), primers of target sequence, Taq polymerase, free dNTPs. Thermocycler goes up to 94-95 degrees C and denatures the DNA strands. Then the thermocycler cools down to ~50-57 degrees C so primers can anneal to the ends of the target sequence. The thermocycler raises its temperature again to 72 degrees C so the Taq polymerase can attach to the primers and extend the DNA. The thermocycler repeats this 20-30 times.

Gel Electrophoresis: ingredients

micropipette, agarose gel and wells, DNA ladder sample, DNA testing samples, buffer solution, electrode powered tray.

Gel Electrophoresis: purpose/application

To measure the length of DNA in base pairs. It can be used in a forensic setting, to determine who is guilty, or even in a setting to see which DNA is most related to each other. These sequences can later be sequenced. This process makes it easier to know which DNA fragments to sequence after performing a test such as PCR.

Gel Electrophoresis: process

Different DNA samples and ladder DNA are put into wells of the ready-made agarose gel with a micropipette. DNA is put on the negatively charged side, so it moves to the opposite end (DNA is naturally negatively charged). The electrically charged box/tray is turned on and the DNA moves across it. The shorter fragments move the farthest while the longer fragments barely move. The gel can be placed under UV light to see they DNA better and the results are analyzed.

Sanger Sequencing: ingredients

Sanger Sequencing: purpose/application

Sanger Sequencing: process

The reaction is heated to 96 degrees C to denature the DNA. The temperature is then lowered to 50 degrees C so the primers can anneal to the ends of the sequence. Temp. is then raised to 60 degrees C so the DNA polymerase can attach to the sequence and add the complementary bases of DNA to the “template” strand. The temp. is then raised again to separate the DNA strands: this process is repeated several times to get many different fragments. Because the DNA polymerase is just as likely to bind an dNTP compared to a ddNTP, there are several fragments of the target DNA, terminated at different places.
these fragments are then run through gel electrophoresis. Since the shorter fragments will have traveled the farthest, the sequence of the template DNA can be determined by reading the complementary base of each band going from the positive to negative direction.
the ddNTP’s are fluorescently labeled, so the sequence can be read by the machine (or not), and the sequence is determined (this was from vid, so check with kestner before test)

Blotting: ingredients

Blotting: purpose/application

to see if a certain piece of DNA or gene is in a large piece of DNA. (usually a gene)

Blotting: process

First you cleave the DNA by exposing it to enzymes that cut it at random areas: this created the fragments. You then run the DNA fragments through gel electrophoresis. You then filter the gel because it is so flimsy. Then the filter is exposed to radiolabeled DNA that is complementary to what you are trying to find. If the radiolabeled DNA anneals to any of the DNA, then the sequence you were wondering whether or not it was present, is indeed in the DNA (after exposing the filter to a x-ray to see where the radio-labeled DNA is).

Different Blotting techniques

southern: presence of DNA, northern: presence of RNA, western: presence of protein (SNOW DROP)

cDNA library: ingredients

cDNA library: purpose/application

To find out the DNA sequence of a protein. From this a DNA library would be made accessible to scientists, clinicians, researchers, etc. For example, if someone has a mutation, it will be easy to find what they have. purpose is to see what genes are expressed in proteins.

cDNA library: process

the mRNA of the protein is isolated and collected. Reverse transcriptase is added and makes complementary DNA (single stranded DNA). DNA polymerase is added to make double stranded DNA. The dsDNA “infects” bacterial plasmids in which the bacteria will rapidly grow and replicate the DNA. The DNA is then isolated and sequenced and added to a database made accessible for everyone.

Microarray: ingredients

“cancer” cell mRNA and normal cell mRNA, microarray chip, complementary mRNA of the different genes tested

Microarray: purpose/application

To detect thousands/lots of genes in cells at the same time. Also used to determine specific mutations.

Microarray: process

The microarray chip contains little wells or holes. These wells can be filled with complementary mRNA of a certain gene. For example, we could have the complementary mRNA for Gene A. The cells’ intracellular contents (with labeled yellow probes) can be added to the well with complementary mRNA. If the gene is upregulated in the cell, then the wells will be bright yellow. Same goes for the downregulated gene. If there’s a lot of the blue (from the blue probes) after it is exposed to the intracellular contents of both cells, then we can figure out if this gene is in the cell or not.

CRISPR: ingredients

Cas9 enzyme, DNA chromosome (containing PAM), guide RNA (gRNA: contains specific sequence of interest)

CRISPR: purpose/application

CRISPR: process

Cas9 enzyme scans DNA for protospacer adjacent motif (PAM) and checks the sequence for a match. If it isn’t a match, then it moves onto another PAM. The Cas9 gRNA hybridizes to matching DNA once it is identified. The enzyme then cuts the DNA.