Download Unit 13: Review Biotechnology Lab

Unit 13: Review
Biotechnology Lab
I. DNA replication
DNA replication is a process in which DNA makes an exact copy. DNA replicates
through a semiconservative method. The chemical direction of replication is 5’
(five prime) to 3’ (three prime).
Examine the deoxyribose (sugar) puzzle piece. Deoxyribose is a pentose, a
monosaccharide containing five carbons. This puzzle piece shows only one of
the carbons, carbon #5.
Continue
Deoxyribose
DNA replication continued
The other four carbons have been superimposed on the puzzle piece to show you
their location.
DNA replication continued
In DNA replication, nitrogenous
bases (N-bases) are
complementary.
H bond
Adenine (A) always binds to
thymine (T) and forms two
hydrogen bonds.
Carbon #1
C
C
Carbon #4
C
C
Carbon #2
Carbon #3
Cytosine (C) always binds to
guanine (G) and forms three
hydrogen bonds.
The double-ringed N-bases are
called purines (A,G) and
single-ringed N-bases are
called pyrimidines (C,T)
Carbon #5
Continue
Continue
DNA replication continued
The DNA molecule is a polymer of
nucleotides.
Recall that a nucleotide is made of three
components,
• a phosphate functional group,
DNA replication continued
DNA replication stages:
1. Unwind the double helix
2. Maintain the strands separate
3. Synthesis of new strands
4. Formation of two dsDNA molecules
•
a pentose (deoxyribose for DNA),
•
and a N-base (A, T, G, or C).
This single-strand of DNA has 5 nucleotides
shown.
Continue
Continue
1
DNA replication continued
1.
DNA replication continued
Unwind the double helix
DNA is a helical, double-stranded molecule. Helicases are enzymes that will unwind
the double helix, preparing it for the next stage.
Continue
2.
Maintain the strands separated
Since the two strands are complementary, they have a tendency to reunite. Singlestrand binding proteins stabilize the two strands.
Continue
The 2 strands are kept separated
DNA strand is unwinding
DNA replication continued
3.
DNA replication continued
4. Form two dsDNA molecules
The result is two dsDNA molecules. Now the cell can continue the process of cell
division (dividing into 2 cells) because it has two copies of the DNA.
Synthesis of new strands
With the exposed N-base sequence, DNA polymerase can add new DNA
nucleotides to the template (in dark blue). The new strand (in light blue) is
synthesized 5’ to 3’.
5’
3’
3’
5’
Continue
5’
3’
3’
5’
5’
3’
3’ 5’
5’ 3’
3’
5’
5’
3’
3’
5’
5’ 3’
Arrows show
chemical direction of
synthesis, 5’ to 3’
5’
3’
5’
3’
5’
3’ 5’
3’
5’
3’
5’
3’
5’
3’
5’ 3’
5’
3’
•
5’
5’
Transcription continued
The other four carbons have been superimposed on the puzzle piece to show you
their location.
Carbon #1
C
C
Continue
5’ 3’
Two dsDNA
molecules
Ribosomal RNA (rRNA) is a component of
ribosomes and involved in protein synthesis
Transfer RNA (tRNA) is the carrier of amino
acids, contains the anticodons
Messenger RNA (mRNA) dictates the amino
acid sequence, contains the codons
The chemical direction of transcription is 5’ to 3’.
Examine the ribose (sugar) molecule of DNA.
Ribose is a pentose, a monosaccharide
containing five carbons.
3’
Continue
Transcription is the synthesis of RNA on a DNA
template. Three major RNA’s are produced:
rRNA, tRNA, and mRNA.
•
5’
3’
II. Transcription
•
3’
Carbon #4
Pentose
C
C
Carbon #2
Carbon #3
Carbon #5
Continue
2
Transcription continued
The RNA molecule is a polymer of
nucleotides.
RNA N-bases are
Adenine (A),
Uracil (U),
Guanine (G),
and Cytosine (C).
Recall that a nucleotide is made of three
components,
• a phosphate functional group,
•
a pentose (ribose for RNA),
•
and a N-base (A, U, G, or C).
Transcription continued
This single-strand of RNA has 6 nucleotides
shown.
Continue
Continue
Transcription continued
Transcription continued
3’
5’
Transcription stages:
3’
dsDNA
1. Unwind the double helix
‘Bubble’
2. Synthesis of a new RNA strand
ssRNA
3. Separation of RNA from DNA, DNA double helix reforms
5’
1. Unwind the double helix
RNA polymerase unwinds the double helix a a ‘bubble’ appears. (The
two blue strands separate.)
2. Synthesis of a new RNA strand (in pink)
RNA polymerase brings in RNA nucleotides complementary to the
DNA template. This action moves from 5’ to 3’ direction.
Continue
Continue
3. Separation of RNA from DNA, DNA double helix reforms
Transcription continued
dsDNA
III. Translation
After the mRNA is synthesized it moves into the cytoplasm and becomes associated
with the ribosome. Translation is the process of having tRNA molecules bring in
correct amino acids which are linked to one another.
DNA template
To translate the message from nucleic acids to amino acids use the table in you lab
manual (The Genetic Code).
RNA
complementary
strand
In transcription if the DNA template reads ………… 5’ ATGC 3’
The RNA complementary strand will read………….. 3’ UACG 5’
Continue
Translation, occurs in 3 steps:
A. Initiation
B. Elongation
C. termination
Continue
3
Translation continued
A. Initiation
The binding of the first tRNA to mRNA. The mRNA codon is 5’ AUG 3’. Because it
initiates translation, this codon is referred to as the start codon. According to the
genetic code, the codon AUG codes for the amino acid Methionine.
The tRNA that carries Methionine has the anticodon, 3’ UAC 5’.Complementary Nbases exist between the codon of the mRNA and the anticodon of the tRNA.
Continue
Translation continued
B. Elongation
The addition of as many amino
acids as needed to build the
polypeptide. Each amino acid is
carried by the tRNA to the mRNA.
Continue
Translation continued
C. Termination
Elongation will continue until a stop signal appears, encoded by a termination
(stop) codon. There are three stop codons, UAA, UAG, UGA. When a stop codon is
encountered, the peptide chain is released, the mRNA and ribosomal subunits
dissociated, and protein synthesis is terminated.
Continue
IV. Isolation of DNA from banana cells
at home lab
The preparation of DNA from any cell type involves the same general steps:
1. breaking open the cell and nuclear membrane (if present),
2. removing proteins and cellular debris from the nucleic acids, and
3. purification of the DNA
This procedure allows for the crude extraction of DNA from banana cells.
Continue
Isolation of DNA continued
Isolation of DNA from banana cells.
Isolation of DNA continued
3. Stir the mixture slowly (avoid creating
foam)
1. A fork is used to mash up a section
(about ¼) of a banana.
Mashed banana
2. In a heat-resistant vessel (i.e. Pyrex)
the mashed banana is combined
with 250 ml deionized water, 25 ml of
a soap solution and 25 ml of meat
tenderizer solution
Mixture
4. Boil the mixture
Meat tenderizer is a saturated solution. Just
add tenderizer to water until it stops going
into solution.
Liquid hand soap solution is 50% water and
50% soap
Continue
Continue
Boiling mixture
4
Isolation of DNA continued
Isolation of DNA continued
5. Strain the mixture through 4 layers of
cheesecloth and collect the filtrate (fluid that
has passed through a filter)
7. Gently layer ethanol over the
filtrate
6. Pipette 10 ml of the filtrate into a test tube
8. DNA precipitates in the
interphase between the
ethanol and filtrate
Ethanol
layer
Examine the appearance of the
extracted DNA.
DNA
Continue
Filtrate
layer
Continue
V. DNA electrophoresis
DNA electrophoresis continued
Restriction endonucleases or restriction enzymes (REs) are enzymes obtained by
bacteria that physically cut DNA. REs are named according to the following
guidelines. The first letter is the same letter as the first letter of the genus name of the
organism from which is was isolated and is italicized. The second and third letters are
the first two letter of the specific epithet and are italicized. The fourth letter, not
italicized, indicates the specific strain of the organism. Roman numerals indicate the
order of discovery.
RE’s recognize a 4- or 6-base pair sequence that is a palindrome and cut the DNA in
the same way every time. REs attach to the DNA at a specific recognition sites,
called restriction sites. Some cut through the complementary strands at the same
position, producing blunt ends. Others cut producing sticky ends.
Blunt ends
Haemophilus aegyptius (bacteria) produces a restriction enzyme, Hae III that
recognizes the palindrome …GGCC… and cuts between the G and the C
producing blunt (or flush) ends.
…G G C C…
…C C G G…
CC…
GG…
+
Sticky ends
Escherichia coli RY13 (bacteria) produces a restriction enzyme, EcoR I that
recognizes the palindrome … GAATTC… and cuts between the G and the A
producing sticky (or staggered) ends.
…G A A T T C…
…C T T A A G…
Continue
…GG
…CC
…G
…GTTAA
+
AATTC…
G…
Continue
DNA electrophoresis continued
The resulting fragments of DNA can
be separated from one another
by electrophoresis.
Electrophoresis is the process of
applying voltage to a solution of
charged molecules (DNA or
proteins).
DNA electrophoresis continued
An electrical field is applied causing the DNA fragments to move from their origin
through the gel toward the positive electrode, since DNA is negatively charged.
Loading the gel
DNA fragments are placed in
wells made on the agarose gel
(similar to gelatin).
one of
the
wells
Continue
Agarose gel
Agarose gel
Continue
5
DNA electrophoresis continued
Wells
The DNA fragments move at different
rates depending on their size. Small
fragments migrate faster than
larger ones. Fragments of the same
size concentrate in one group
forming a band (thin line) on the
gel.
The gel is removed from the chamber
and stained.
Examine the stained gel. Remember
that the DNA fragments all start in
the well and travel toward the right
side, from the cathode(-) to the
anode (+) end.
End of Lab
Review ☺
-
+
6