Techniques for Producing and Analyzing DNA

recombinant DNA

DNA created in a lab by combining DNA fragments from different organisms (even different species)

done to give an organism a new trait

What enzymes are involved in recombinant DNA

restriction enzymes or endonucleases

restriction enzymes purpose

cut viral DNA to protect bacteria from infections

what are restriction enzymes/endonucleases

Enzymes that cleaves double-stranded DNA at specific internal sequences, not at the ends

allows scientists to precisely remove or insert genes without damaging the rest of the DNA

sticky ends

overhanging single-stranded pieces of DNA created when restriction enzymes cut DNA in a zigzag or staggered way

they bind to other DNA fragments with complementary sticky ends

How do restriction endonucleases work?

bind to a specific sequence (restriction site) and make cuts that create restriction fragments

after the cut, sticky ends help fragments pair together to combine DNA with other sticky ends that have complementary base sequences

DNA ligase is added to fuse the strands together to create a smooth piece of recombinant DNA

gene cloning

using recombinant DNA to make identical copies of a specific gene using a host organism

allow the study of a specific gene

common host organisms

bacteria, yeast, insect cells

easy to work with, cheap to grow

plasmid and importance

small circular piece of DNA in the host that contains the actual gene of interest to carry and copy

actual DNA molecule that holds the gene

vector

gene of interest being inserted into plasmid

lac Z gene

Shows which plasmids got the gene inserted (some will absorbé the plasmid without the gene because it was resealed)

interested gene creates an enzyme that breaks down X-gal

when x-gal is broken down, produces a blue colour

What do we add to the plasmid to track the cloning process (types of trackers)?

lac Z gene

An ampicillin resistance gene

A restriction site where the gene is inserted

ampicillin resistance gene

Shows if a bacterium took up any plasmid at all

the interested gene makes the bacteria resistant to the antibiotic ampicillin

Only bacteria that has plasmid will survive when grown on an ampicillin plate

why is restriction site needed to track cloning

how we control exactly where the new DNA goes, so we know what we’re disrupting

How do you know which bacteria took up the gene of interest?

plate the bacteria on a dish with ampicillin and X-gal

White colonies = have the gene of interest (lac Z was broken)

Blue colonies = have a working lac Z gene (no gene inserted)

No colonies = those bacteria got killed by the ampicillin (no plasmid)

what happens to the white colonies

grown separately to determine which ones contain the actual gene of interest, some may contain genes that were inserted that were not the one being studied

isolate the gene and grow them in large numbers

Polymerase chain reaction

amplify/copy millions of copies of a specific DNA segment without recombinant DNA or vectors

step 1 of PCR (denaturation)

sample is heated up to 95 degrees to break H bonds of DNA

it unwinds the helix into two single strands

just like in DNA replication, must be separated before copying occurs

step 2 of PCR (annealing)

temperature is lowered to 55 degrees so the primers (short DNA sequences) can attach/anneal to the single-stranded DNA at the right spot

primers tell the enzyme where to start copying

why is temperature lowered for the annealing step

strands are too hot for base pairing to occur

to allow H bonds form again at ideal temp

step 3 of PCR (extension)

the temp is raised slightly to 72 degrees for Taq polymerase’ optimal condition

Taq adds new nucleotides to each primer to create two new double-stranded DNA molecules from the original one

taq polymerase and why it is used in PCR

a heat-resistant DNA-building enzyme

it can survive high heat without denaturing (unlike normal enzymes) because PCR has to be repeated multiple times (constantly exposed to heat)

step 4 of PCR

DNA is taken through this PCR cycle several times to produce a mass amount of copies

doubles the copy every cycle

What is gel electrophoresis used for?

separate fragments of DNA (negatively charged) by size through a gel using an electric field

will create a pattern on the gel that can be compared to known DNA samples

how does gel electrophoresis help with the analyzation of DNA

helps scientists see the differences in different fragment samples