Extra resources Flashcards
What is DNA?
The molecule of inheritance in living organisms.
Who created the modern discipline of geentics?
Gregor Mendel.
What was Mendel’s experiment?
Breeding plants and animals.
How did Mendel do his experiments?
With pea plants in his monastery garden.
What was Mendel doing with peas in his experiment?
Observing several plants’ characteristics.
What were some of the characteristics Mendel was observing in his experiments?
Seed colour, shape, flower location, colour, pod colour, shape, height.
How was Mendel breeding the peas in his experiment?
Breeding standard generation twice –> 3 generations of peas.
What did the standard deviation of Mendel’s experiment have?
One homozygous plant dominant.
One homozygous recessive.
What did the first generation of Mendel’s experiment with peas have?
All peas heterozygous.
What did the second generation of peas in Mendel’s experiment have?
- homozygous dominant.
- homozygous recessive.
3 & 4. heterozygous.
What were the generalizations of Mendel’s experiment?
- Mendel’s Law of Segregation: every organism has 2 alleles of a gene.
- Mendel’s Law of Independent Assortment: alleles are passed on independently of each other.
What did Mendel proposed?
Dominant traits mask recessive traits.
Where else is Mendel’s experiment applied?
On individual genes.
What experiments did August Weismann do?
With mice.
What did mice experiments of Weismann show?
Traits inherited by organisms during lifetime did not pass on to offspring.
What theory did Weismann proposed?
The germ plasm theory.
What did the germ plasm theory state?
Hereditary information stored in egg and sperm cells of eukaryotic organisms.
What were proteins responsible for?
Genetic inheritance.
What did Johann Miescher do in 1869?
Isolated DNA from white blood cells.
What did Albrecht Kossel do in 1878?
Isolated nucleic acids of DNA and RNA.
What were the 3 choices presented for genetic inheritance?
- DNA.
- RNA.
- Proteins.
What was Frederick Griffith investigating?
How organisms pass on genetic information to their offspring.
With what was Griffith working?
Streptococcus pneumoniae bacteria.
With which types of Streptococcus pneumoniae did Griffith work?
Rough.
Smooth.
What was the differences between rough and smooth Streptococcus pneumoniae bacteria that Griffith worked with?
Smooth: covered with saccharide layer, harder to detect for white blood cells.
Rough: easily killed.
Where did Griffith test bacteria?
On mice.
What did Griffith find about testing bacteria on mice?
Mice exposed to smooth bacteria –> died.
Mice exposed to rough bacteria –> survived.
What else did Griffith do with the bacteria?
Exposed them to heat.
What did Griffith with bacteria exposed to heat?
Exposed them to mice.
What did Griffith find when exposed heat bacteria to mice?
Heat stressed rough bacteria –> no negative effects on mice.
Heat stressed smooth bacteria –> died.
What did Oswald Aver, Colin MacLeod and Maclyn McCarty do experiments for?
To show if DNA, proteins, or RNA acted as the molecule of genetic transmission.
What did Avery’s team do?
Isolated DNA, RNA, Proteins from cells of strep pneumoniae as Griffith –> treated with enzyme to break down one type of molecules –> solutions exposed to rough strains –> injected to mice –> not died.
What was the conclusion of Avery’s team?
DNA is the molecule of genetic inheritance.
What experiments did Hershey and Chase do?
Experiments on bacteriophages, viruses that infected bacteria –> proliferate themselves –> exposing bacteriophages to radioactive isotopes of phosphorus and sulphur –> incorporated in virus’ DNA or proteins –> sulphur found in one of each not in both –> phosphorus into DNA & sulphur into proteins –> check which bacteria inherited radioactive indicators –> bacteria with phosphorus became radioactive –> bacteria with sulphur did not.
What was the conclusion of Hershey and Chase experiment?
Virus molecule of inheritance is DNA.
What did Edward Tatum and George Beadle show in 1940?
DNA genes are directly responsible for creation of cellular proteins.
What did Tatum and Beadle use for experimental subject?
Neurospora crassa = bread mould.
How did Beadle and Tatum use Neurospora crassa?
Spores of Neurospora lighted –> mutate genes –> crossed mutated spores with normal ones –> mutant offspring.
What happened in Beadle and Tatum’s experiment with Neurospora?
Normal spores grew on regular growth medium.
Mutant offspring required addition of arginine to grow on medium.
Why did the mutant offspring require addition of arginine to grow on medium?
Mutated genes are coded for protein that produces arginine amino acid.
What did Levene identify in 1919?
Deoxyribose saccharide phosphate group.
Nucleic acids DNA is made up of.
What did Levene propose?
DNA consists of nucleotides linked by phosphorus groups.
DNA is very short.
What did Elsif propose?
DNA structure = 2 strands = template for reproduction.
What did Jean Brock show in 1933?
DNA is organised in chromosomes.
What did William Astbury provide in 1937?
X-ray diffraction images –> DNA regular structure.
What else did Chargaff use?
X-ray chromatography.
What did Chargaff find with X-ray chromatography?
Relative amount of each nucleic acid in DNA.
Cytosine = Guanine. Adenine = Thymine.
What did James Watson and Francis Crick create in 1953?
Double helix model of Deoxyribonucleic acid.
What is important about Francis and Watson model?
Accepted DNA model today.
On what was Watson and Crick’s model based?
On X-ray diffraction by Rosalind Franklin.
What did Watson and Crick find with Rosalind’s model?
2 strands of DNA backbone = identical.
On what is molecular biology based?
Double helix model of DNA with nucleic acids in centre and phosphate + deoxyribose groups chains.
What did Matthew Mason and Franklin Stoll show in 1958?
DNA replicates semi-conservatively = DNA replicates –> half of new strand from parent strand + half newly made up.
What did Mason and Stoll do?
Bacterial DNA –> took light and heavy isotopes of Nitrogen –> centrifuged DNA after replication –> separating it by consistency.
How is DNA used?
Template –> transcription –> creates RNA –> template –> translation –> creates proteins.
What happens during transcription?
mRNA lives cell nucleus –> enters cytoplasm –> binds ribosome –> 3 nucleic acid of RNA interpreted as a codon –> correspond to an amino acid –> amino acids go to ribosome by tRNAs –> synthesized into primary protein structures –> fold in functioning proteins.
What can we find inside DNA?
Double-stranded DNA = 2 strands braid –> form double helix.
What is the most common form of a DNA double helix?
B-form DNA.
What is each strand of DNA?
A polynucleotide made up of many individual units, nucleotides.
What does a nucleotide have?
3 components:
- 5-C sugar.
- Phosphate.
- One possible base (A, G, T, C).
Where is the nitrogenous base always attached?
At 1-C of sugar.
Where is the phosphate attached?
At 5-C of sugar on 1 nucleotide and 3-C sugar on previous nucleotide.
How is the sugar of DNA called?
Deoxyribose.
Why is the sugar called deoxyribose?
Missing a OH group at 2-C present in ribose.
How are nucleotides in DNA, called?
Deoxynucleotides.
How are nucleotides bind to each other in a DNA strand?
By phosphodiester bonds.
What do phosphate group + sugar make?
DNA backbone.
What is the direction of the DNA strands?
Top: 5’- 3’.
Bottom: 3’- 5’.
How can we see clearly the structure of DNA?
Unwinding.
Flattening double helix.
How do the 2 DNA strands interact with each other?
Through non-covalent hydrogen bonds between bases..
What does each base of DNA structure form?
Hydrogen bonds with the complementary base on the opposite strand.
What is a base pair?
A unit of 2 bases connected with each other through hydrogen bonds.
How are bases connected?
A = T (2 H bonds) G = C (3 H bonds)
How are thymine and cytosine called?
Pyrimidines = single ring structure.
How are adenine and guanine called?
Purines = double rings.
Is the geometry of bases in DNA the same no matter what base is?
Yes.
Why can not other bases form base pairs?
No geometry.
Not strong H bonds are formed.
Disturb helix.
How many base pairs occur in each turn of the DNA helix?
10.
What does the structure of bases and connection through H bonds forms?
Stable structure of DNA.
When are the pi-pi interactions formed?
When aromatic rings of bases stack next to each other –> share electron probabilities.
What else is it formed from the double helix structure of DNA?
2 spaces:
- Major grooves.
- Minor grooves.
How do grooves in DNA act?
Base pair recognition.
Binding sites for proteins.
What does the major groove of DNA contain?
Base pair specific information.
What is the minor groove of DNA?
Base pair nonspecific.
Why are major and minor grooves of DNA different?
Due to different acceptors and donor which proteins can interact with.
In which ways can DNA be acted?
- Sequence specific.
2. Non-sequence specific manner.
What is the cell?
The basic unit of all living tissue.
Where can nucleus be found and what does it contain?
In human cells.
The genome.
Into what is the genome split in humans?
To 23 chromosome pairs.
What does each chromosome contain?
Long strand of DNA –> tightly packaged around proteins = histones.
What does occur within DNA?
Sectors = genes.
What do genes contain?
Instructions to make proteins.
What happens when a gene is switched on?
Enzyme RNA polymerase –> attaches gene’s start.
What does RNA polymerase do?
Moves along DNA –> makes mRNA strand, in nucleus.
What does DNA do while RNA polymerase creates mRNA?
Codes bases of mRNA ON new strand.
Can the mRNA used as a template once it is transcribed from DNA?
No.
It needs to be processed.
How is mRNA processed to used as a template?
Removing and adding RNA sections.
Where does mRNA go once it is processed?
Out of nucleus –> into cytoplasm.
What happens to mRNA once it enters cytoplasm?
Ribosomes bind to it –> read code on it –> produce amino acids chain.
How many different types of amino acid occur?
20.
What do tRNAs do?
Transfer amino acids to ribosome as each triplet on mRNA is read.
How is mRNA read?
3 bases at a time.
Where are amino acids from tRNA added?
To a growing chain of amino acids.
What happens once the last amino acid is added?
Chain falls to a 3D shape –> form protein.
What is transcription and translation?
Collective process where genetic code read by enzymes –> produces proteins in organism.
What is a chromosome?
A very long molecule.
Of what does a chromosome consist?
Millions of base pairs.
Are all of the parts of chromosome special?
No.
What parts of chromosomes are special?
Genes.
What do genes do?
Code for different things.
How long is a gene in humans?
10-50 thousand base pairs.
How long can the longest chromosome be?
2.5 million base pairs.
What happens when a gene is expressed?
A specific protein is produced?
What is transcription?
Process of enzymes use one of strands of DNA in gene as template –> produce mRNA.
How does transcription occur?
Enzyme RNA Polymerase + proteins transcription factors bind to specific sequence promoter –> 2 strands apart –> 1 strand = template/antisense strand –> used to generate mRNA –> other strand = nontemplate strand/sense strand.
Does RNA polymerase need a primer?
No.
What does RNA Polymerase do?
Moves along DNA –> elongation –> synthesizes mRNA as it goes.
Reading antisense strand from 3’- 5’ –> generating mRNA 5’ - 3’.
What is the difference between the newly synthesized mRNA and the template DNA strands?
DNA: deoxyribose sugar, A-C-G-T
RNA: ribose sugar, A-C-G-U
How many bases of DNA are exposed at a time?
10-20.
What happens to DNA strands after mRNA is synthesized?
Wraps back up.
What happens once RNA polymerase reaches the end of the gene?
Termination occurs =
Enzyme detaches from gene –> DNA returns to original state –> mRNA produces.
Where does translation occur?
In ribosome.
What happens during translation?
mRNA acts as a code for a specific protein –> 3 codons on mRNA –> code for a specific anti-codon –> carried by tRNA –> covalently linked to amino acid.
What is the reading frame?
Nucleotides into codons on mRNA strand.
How many possible codons occur?
64 (3 on 4).
What is the strange thing in the universal genetic code?
Multiple codons code same amino acid.
To what does each codon correspond?
To a particular amino acid.
Which is the start codon?
AUG.
What does AUG codon do?
Initiates translation –> codes for Methionine.
Which are the stop codons?
UAA
UAG
UGA
What do stop codons do?
Terminate (finish) translation.
Where does the small ribosomal subunit bind?
To mRNA.
To tRNA.
What does the large ribosomal subunit do?
Joins after first tRNA binds small ribosomal subunit –> completes translation initiation.
What happens after the large ribosomal subunit binds mRNA?
Second tRNA brings second anti-codon to mRNA (codon) –> second amino acid binds first amino acid –> first tRNA leaves –> process continues along mRNA sequence.
What happens in the end of translation process?
Polypeptide chain grows.
When does translation stop?
When a stop codon occur –> completed polypeptide flies away –> enters cell/organelle for modification.
How does DNA –> transcribed –> mRNA –> translated –> protein?
Obeying to base pairing in nucleic acids.
What do proteins do?
Make the most of us = tissues, organs, receptors, enzymes.
What does DNA do?
Carries genetic code for living organisms.
What is protein synthesis?
Process of making proteins.
Which steps include protein synthesis?
- Transcription.
2. Translation.
What is transcritpion?
Copying a single DNA to mRNA.
What is Translation?
Taking mRNA strand –> use it –> produce a protein.
What does occur inside almost every cell?
Nucleus.
What occurs in nucleus?
All genetic material of each cell in DNA form.
Why do we save DNA?
- Essential for life.
- Controls what cells do.
- Contains thousand genes.
What are genes?
Smaller DNA sections with specific sequences –> code for specific amino acids sequences –> combined –> form a protein.
How can we make a protein?
Specific sequence of gene –> read by ribosomes.
Where do ribosomes occur?
Outside nucleus.
Is DNA big?
Yes.
Why do we need to make a copy of a gene to use it?
Because DNA is so big –> cannot leave nucleus.
What do we actually copy?
A single gene.
Not the whole DNA strand.
Can the copy of gene leave the nucleus?
Yes.
Small enough.
Where does the copy of the gene go?
To the ribosome.
What is the copy of the gene?
mRNA = messenger RNA.
How is the structure of mRNA?
Mostly similar to DNA.
Differences:
Much shorter.
Only a single strand.
Uracil base not Thymine.
How do we see DNA normally in nucleus?
2 strands fold into a helix.
What is a simplified version of DNA?
Unwind.
How does Transcription process start?
With RNA Polymerase enzyme.
Where does RNA Polymerase bind?
To DNA right before the gene to be coded, starts.
Where do the 2 strands of DNA separate apat?
Just ahead of RNA Polymerase.
What happens when the 2 strands of DNA separate apart?
Bases are exposed.
What does RNA Polymerase do once the 2 strands separate?
Moves along DNA strand –> read bases one by one –> use them –> make mRNA.
What will always mRNA bases be?
Complementary to DNA bases.
With what will the DNA bases bind with an mRNA base?
C, G, T, A?
C = G G = C T = A A = U
With what are all of the thymine on DNA replaced on mRNA?
With Uracil.
What does the DNA strand do while RNA Polymerase is moving along the strand and synthesizing mRNA?
Opens up to the right and closes up from the left.
How much of the DNA is exposed at a time?
Only a small section of it at a time.
What do RNA Polymerase and DNA do once mRNA is fully synthesized?
RNA Pol: Detaches from DNA.
DNA: closes back up.
Where does mRNA go at the end of transcription?
Leaves nucleus –> heads to ribosome.
How is the DNA strand where RNA Polymerase moved along, called?
Template Strand.
What is the template strand of DNA sued for?
To make mRNA.
Once mRNA moves to the ribosome what happens?
Undergoes translation –> produces protein.
How is each group of 3 bases for both DNA and RNA, called?
Codon/Triplet.
For what does triplet from DNA/RNA code?
A specific amino acid.
For which amino acid does the triplet ‘AGU’ code?
Serine.
‘CCA’?
Proline.
How does the translation process start?
mRNA and ribosome bind together.
Where are amino acids bind?
At tRNA = transfer RNA.
What do tRNA molecules have?
Anti-codon on bottom.
Amino acid on top.
What is an anti-codon?
3 bases complimentary to the 3 bases on mRNA.
What do the 3 bases on mRNA do?
Code for amino acid that tRNA carries.
To what is each type of tRNA specific?
To a particular triplet on mRNA.
Which anti-codon and amino acid does the codon ‘AGU’ code?
‘UCA’: anti-codon
‘serine’: amino acid.
What do linked amino acids build?
A chain of amino acids.
What does the ribosome do once the amino acids are linked together?
Moves along mRNA slightly, to the next codon.
What happens once ribosome moves to the next codon?
First tRNA is detached –> amino acid is left behind linked to the next amino acid.
What happens to the amino acid chain once is detached from the ribosome and mRNA?
Folds up itself –> forms a protein.
What does synthesis mean?
Make something.
Where are enzymes involved?
In transport.
Structure.
Enzymes to make materials.
Protecting the body.
Why is protein synthesis essential?
To live.
When does protein synthesis occur?
All the time.
Where is our DNA?
In nucleus.
What is RNA?
A nucleic acid like DNA, with few differences.
Which of the protein synthesis process comes first?
Transcription.
In what to transcribe DNA in transcription?
To a message.
Where does transcription occur?
In nucleus.
Of what does messenger RNA consist?
A message made from RNA based on the DNA.
What is the good thing about the mRNA in eukaryote organisms?
Gets out of nucleus.
In cytoplasm.
Attach ribosomes.
What do ribosomes make and where?
Proteins in translation.
Of what are ribosomes made?
rRNA = ribosomal RNA.
Where do we have tRNAs available?
In cytoplasm.
What are tRNAs?
Transfer RNAs.
What do tRNAs carry?
An amino acid.
What is an amino acid?
A monomer of a protein.
A building block for a protein.
Why is mRNA important in translation process?
Directs which tRNAs come in –> which amino acids are transferred.
What are tRNAs looking for?
Complementary bases.
What do tRNAs do once they find their complementary base on mRNA?
Transfer their amino acid.
What do tRNAs read?
Bases on mRNAs as 3, in triplets = codon.
What do we use the codon chart for?
To find which amino acid each mRNA codon will code for.
Which is most of the times/normally the first amino acid in proteins?
Methionine = AUG.
How do tRNAs work in translation process?
Bring one amino acid –> link it –> leave –> bring another amino acid.
How are amino acids linked to build a protein (polypeptide)?
With a peptide bond.
What do stop codons do?
Indicate that protein building is finished.
Do stop codons code for an amino acid?
No.
What is the result of translation?
Build a chain of amino acids brought in based on mRNA coding, complementary to DNA.
Which molecule is the director of the entire protein building?
DNA.
Which molecules help in protein synthesis?
DNA
mRNA
rRNA
tRNA
What happens to the protein after it is detached from the mRNA?
Enters a cell/organelle –> folding –> modification –> transported: depends on protein structure & function.
Of what can proteins consist?
1 or more polypeptide chains.
What is the structure of DNA molecule?
Spiralling chain-like molecule.
How many different types of nucleotides does DNA have?
4: A, C, G, T.
What is a gene?
A specific sequence of DNA, of As, Cs, Ts, and Gs that codes for something.
What proteins do in our body?
Coded by genes –> interact with other proteins and molecule –> make living cells.
What do cells make?
Tissues.
What do tissues make?
Organs.
What do organs make?
Entire living creatures.
Why do individuals have different traits?
Due to mutations in their genetic code.
What do differences in humans’ genetic code cause?
Changes in specific protein shape & its function.
What can mutations change?
When/how much of a particular protein can be produced.
What is a chromosome?
An entire chain of DNA + group of stabilizing proteins.
Of what do chromosomes consist?
Collection of histones wrapped with a string-like structure.
What are the histones wrapped with a string-like structure?
The chain of DNA.
How is the chain of DNA described?
Extremely long.
With million nucleotides and hundred genes.
How are chromosomes packaged when cells are reproducing?
Tightly.
How do chromosomes exist for most of their lives?
Loose.
Noodle-like structure
With other chromosomes.
Where do chromosomes exist for most of their lives?
Inside nucleus/centre of each cell.
What is our genome?
The entire collection of genes that makes us who we are.
Of how many pairs of chromosomes does our genome consist?
23 pairs.
What does each cell of our, with few exception, contain?
Our body’s full copy of chromosomes.
Our entire genetic code.
What genes do our eye balls use?
Only eye ball cell genes.
Rest are turned off.
What genes does each organ use?
Organ cells only.
Rest is turned off.
From where does 1 member form each pair of chromosomes come?
1 from mother + 1 from father.
What can we say about 2 chromosome pairs if examined together?
They mostly have the exact same genes, at the exact same locations.
What would we find if we were testing the genetic code of 2 chromosome pairs?
Slight sequence variations between them.
Why do chromosome pairs have differences in genetic codes?
Due to mutations.
When do mutations happen?
Happened long ago –> passed down from parent to child : for many generations.
Or
Unique –> over development –> as we mature.
What mutations represent?
Brand new genetic information.
How many unique mutations does an average person have?
50-200.
Are all the mutation that happen to our genetic code bad?
No.
Why are not all the mutations bad?
Because they just make us different from each other.
What are the mutations?
Sequences of DNA we have never seen before in history.
What happens to chromosomes when it comes to reproduction?
Chromosomes –> copied –> condensed –> cell prepares for reproduction –> cells splits in 2.
Why is the DNA tightly packed up in our body?
To fit into the nucleus of every cell.
How does the process of packing tightly the DNA start?
A nucleosome is formed when eight separate histones attach DNA.
What is the nucleosome?
Combined tight loop of DNA + protein.
How are nucleosomes packed with DNA?
Multiple nucleosomes –> coiled together –> stack on top of each other.
What is the end result of packed nucleosomes all together?
A fibre of packed nucleosomes = chromatin.
How thick is the fibre of nucleosomes?
30nm.
What happens to the nucleosome fibre?
Nucleosome fibre –> looped –> further packaged with other proteins.
How much of DNA fits into the nucleus of each cell in our body?
6 feet.
How is nucleus described?
A really small object.
How many nuclei can fit on the tip of a needle?
10 thousand.
What is the end result of the DNA packaging process?
DNA –> tightly packed into chromosomes.
How can we see chromosomes?
Through a microscope.
Are chromosomes always present?
No.
When do chromosomes form?
Only when cells are dividing.
What happens at the end of cell division?
DNA –> less highly organized.
What does ‘DNA Replication’ mean?
Making more DNA.
Where does DNA Replication occur in eukarytic cells?
In nucleus.
Do all cells do DNA replication even if they are eukaryotic or prokaryotic?
Yes.
When does DNA replication occur?
Before cell division.
Why does DNA replication occur before cell division, in a cell?
So daughter cells can also get a copy of DNA.
When does DNA Replication happen, specifically in a eukaryotic cell?
Before mitosis/meiosis.
In interphase.
What are many of the key players in DNA Replication?
Enzymes.
How can we recognise if something is an enzyme in biology?
If it ends with -ase.
What do enzymes do?
Speed up reactions.
Build up.
Break down items they act on.
How is ‘helicase’ characterised?
The unzipping enzyme.
What does helicase do?
Unzipping the 2 strands of DNA –> breaks through H bonds which hold DNA bases together.
How is ‘DNA Polymerase’ characterised?
The Builder.
What does DNA Polymerase do?
Replicates DNA molecules –> build new strand of DNA.
How is ‘Primase’ characterised?
The initializer.
Why do we need the primer?
Because DNA Polymerase can not figure out where to get started without it.
How can we get the primer?
Primase makes the primer.
Of what is the primer made?
RNA.
How is ‘Ligase’ characterised?
The gluer.
What does Ligase do?
Helps glue DNA fragments together.
Where does DNA replication start?
At a certain part called the origin.
How is the origin identified?
By certain DNA sequences.
What happens at the origin?
Helicase –> comes –> unwinds DNA.
Which proteins help the DNA strands to not zip back together once they unwind by helicase?
SSB Proteins.
What are the SSB Proteins?
Single stranded binding proteins.
What do SSB Proteins do?
Bind to the DNA strands –> keep them separated.
What does ‘topoisomerase’ do?
Keeps the DNA from supercoiling.
What is supercoiling?
Something that needs to be controlled during DNA replication.
What does supercoiling need to be controlled?
Because it can involve over-winding of DNA and we need separated strands for next steps.
What happens after 2 strands of DNA unwind?
Primase comes –> makes RNA primers on both strands.
What are the 2 DNA strands?
Antiparallel.
What does ‘Antiparallel strands of DNA’ mean?
They do not go in the same direction.
What is the sugar of DNA part of?
The DNA backbone.
What does the sugar of DNA have?
Carbons.
How are the carbons on the sugar numbered?
Right after the oxygen in a clockwise direction.
How are the 5 Carbons in the sugar placed?
4 carbons in the sugar and the 5th outside of the ring structure.
How do we count the carbons on the antiparallel sugar?
The same way, after the oxygen anticlockwise.
How do the DNA strands run?
One: 5’ - 3’ .
Other one: 3’ - 5’.
Based on how the carbons are numbered.
How is the top original strand of DNA labelled?
3’ - 5’ .
How is the bottom original strand of the DNA labelled?
5’ - 3’ .
What happens after primase comes?
DNA Polymerase comes –> building the new strands on each original strand.
In what direction can DNA Polymerase build the new strand only?
5’ - 3’.
Where does DNA Polymerase add new bases on the new strand?
On the 3’ prime end.
How does DNA Polymerase keeps building the new strand?
As DNA unwinds.
Which is the Leading strand?
The new building strand with orientation 5’ - 3’.
How is the new strand known?
As the Lagging strand with orientation 3’ - 5’.
What must happen on the lagging strand?
Primers placed to help DNA Polymerase build the strand.
What occurs on the lagging strand?
Fragments.
How are the fragments on the lagging strand known?
‘Okazaki Fragments’.
What do the primers do to the ‘Okazaki Fragments’ on the lagging strand?
Replace them with DNA bases.
What does happen after Okazaki fragments appear?
Ligase seals Okazaki fragments together.
What do we have at the end of DNA replication?
2 identical double helix DNA molecules from 1 original double helix DNA molecule.
How do we call the DNA replication?
‘Semi-conservative’.
Why do we call DNA Replication ‘Semi-conservative’?
The 2 copies contain 1 old original strand + 1 newly made strand.
What can happen if DNA Polymerase matches the wrong DNA bases?
We will get an incorrectly coded gene.
Where could an incorrectly coded gene end up?
In an incorrect protein/no protein.
What does DNA Polymerase have?
Proofreading ability.
What is the advantage of the proofreading ability of the DNA Polymerase?
It rarely makes a mistake.
Where did the understanding of DNA replication led?
To lifesaving medical treatments which can stop DNA replication in harmful cells of pathogenic bacteria/human cancer cells.
How are the 2 DNA strands characterised?
Complementary.
What does ‘Complementary DNA strands’ mean?
Where there is an A there is a T in the opposite strand, where there is a G there is a C in the opposite strand.
What does the direction of each DNA strand show?
How each strand is replicated.
What is the result of the helicase unzipping the DNA strands?
Forms replication fork.
What does each of the separated DNA strands provide?
A template for creating a new DNA strand.
What does the primase do?
Makes a small piece of RNA, the primer.
What does the primer do?
It marks the starting point for the construction of the new strand of DNA.
Where does DNA Polymerase bind?
To the primer.
How is the new leading strand of DNA made?
Continuously.
Can the other new lagging strand of DNA be made continuously?
No.
Why does the lagging strand not made continuously?
Because it runs in the opposite direction 3’ - 5’.
How does the DNA Polymerase make the lagging strabd?
In a series of small chunks, Okazaki fragments.
How does each Okazaki fragment start?
With an RNA primer.
What happens after a primer comes to the lagging strabd?
DNA Polymerase adds DNA bases in 5’- 3’ direction.
How often does a primer been added to the lagging strand?
On primer is added –> DNA polymerase adds a series of DNA bases –> another primer to a further point is added…
What happens once all the DNA strand is made?
Exonuclease removes –> RNA primer from both strand of DNA.
What happens once the primers are removed from DNA?
Another DNA Polymerase fills the gaps left behind with DNA.
What happens after DNA polymerase finishes filling the gaps with DNA?
DNA ligase –> seals up –> DNA fragments in both strands –> form continuous double strand (2).
How is the old strand of DNA called?
Conserved.
For what can gel electrophoresis used?
To separate molecules based on their size.
Where is gel electrophoresis sueful?
In DNA.
What can we find if we zoom in DNA?
A nucleotide.
What is the nucleotide?
A building block of DNA.
What are the phosphates in th enucleotides?
Negatively charged.
What does the negatively charged phosphate in the nucleotide, gives the whole DNA?
A negative charge.
Where does gel electrophoresis rely?
On the negatively charged DNA molecules.
What is the point of the gel electrophoresis machine?
To have an electrical charge running through a gel.
Of what is the gel in gel electrophoresis machine made?
Agarose.
What is agarose?
A polysaccharide polymer.
What are the polysaccharides?
Carbohydrates.
Where from does agarose come?
Seaweed.
What does the agarose gel do?
Lets DNA molecules travel in it.
What does the one end of the gel have?
Holes = wells.
What are the wells in gel electrophoresis?
Where DNA is placed into.
What is the area of the gel where the wells are?
Negatively charged.
What is the area of the other end of the gel?
Positively charged.
Towards where does DNA travel?
From negative area to positive area.
What do we use when we analyse DNA in electrophoresis?
Restriction enzymes.
Why do we use restriction enzymes to analyse DNA in electrophoresis?
To cut DNA up into tiny pieces.
What is the ability of restriction enzymes?
Cut up DNA in very specific areas, based on specific DNA bases.
How important are restriction enzymes in biotechnology?
Very useful.
How can we I compare DNA from a baby guppy and a mother gubby?
Use same restriction enzymes in both DNA samples.
Why should I use the same restriction enzyme to compare a baby DNA and a mother DNA?
To cut DNA at same identification points in DNA samples.
What is the result of adding restriction enzymes to DNA samples?
Pieces cut in samples –> have different size.
Why do the cut pieces of DNA have different sizes even if the same restriction enzymes have been used?
Because DNA of samples had differences in DNA bases sequence.
What happens after the samples of DNA are cut into pieces?
Samples –> loaded into –> gel.
What do we do after we load the samples in the gel?
Turn on the gel electrophoresis machine.
What happens once the machine is on?
DNA runs through gel towards positive side.
What will happen as DNA pieces move towards the positive side in the gel?
Some pieces move faster.
Which DNA pieces move faster in the gel?
Shorter pieces.
Why do longer DNA pieces move slower in the gel?
Because they have a higher molecular weight.
They take more time to go across the gel.
What is the result image in the gel electrophoresis?
Fragments are spread out.
Longer pieces = closer to well.
Shorter pieces = closer to gel’s opposite site.
How are the DNA pieces in the gel, called?
DNA bands.
How can we see the DNA bands in the gel?
Stain gel.
View it under UV light.
If we test organisms that are not clones, will their DNA bands be identical?
No.
How can we compare the DNA bands?
Between each other.
Between babies and mothers.
With other mother.
See how similar they are.
What is a DNA Ladder?
A sample with known fragment sizes.
How can we use the DNA ladder?
Run it in gel –> find the known DNA bands –> use it as reference to compare the other fragments.
How can we make sure we are closer to the value of the DNA ladder bands?
We can use a ‘Semi-Log Graph’.
Why do we use gel electrophoresis?
To show relatedness between different species –> classify organisms.
How else can we use gel electrophoresis?
Part of DNA fingerprinting.
What is DNA fingerprinting?
Identification of someone’s DNA.
Where is DNA fingerprinting useful?
When solving a mystery in a crime scene.
How can we solve a mystery from a crime scene with DNA fingerprinting?
Sample from crime scene –> electrophoresis –> take results –> isolate genes of interest with southern blotting –> compare it to suspect DNA –> see likelihood of match
Where is gel electrophoresis useful?
In biotechnology.
What do we put in one of the lanes of gel electrophoresis?
A DNA ladder with DNA fragments of known sizes.
What do we put in another line of gel electrophoresis test?
A DNA sample with DNA fragment of unknown size.
What do we want to find in out gel electrophoresis test?
The size of the unknown band of our DNA sample.
What must we do once we image the gel?
Label it up.
How do we label our test in gel electrophoresis?
Put lane numbers on top.
Label DNA ladder.
What do we do after labelling our test and DNA ladder?
Start collecting data from gel.
Why do we want to start collecting data from the gel?
To plot a calibration curve.
What do we know in our test?
The size of the DNA fragments in the DNA ladder.
Where do we put our known values?
Into a table.
How do we treat the data we know in the table?
Convert size numbers of DNA ladder to the log value.
Why do to convert the sizes to the log value?
Because when we plot the graph we will get a straight line.
What do we measure once we do our table and convert the values to the log?
The distances that the bands in DNA ladder moved.
How do we measure the bands of the DNA ladder?
From centre of well to the centre of band.
What else do we measure once we measure all the distances of all the bands in the DNA ladder?
The distances of unknown bands.
What do we do once we finish measuring the distances of the unknown bands?
Plot the graph.
Where do we plot the graph?
Using a graph paper.
What do we put on the x-axis of our graph?
The thing we know = log base pairs.
What do we put on y-axis of the graph?
The distance travelled in millimetres.
What is important to do on the graph?
Label the axis = Distance (mm), Log10 (Base Pairs).
Where do we always put the thing we know?
On the bottom of the graph.
What do we after we label the axis??
Plot the points.
What do we do after we plot the points on the graph?
Add line of best fit.
Why do we add a line of best fit with the points on the graph?
For the points to be equally balanced on either side.
What do we finally do?
Give a title to the graph = Graph of Distance (mm) against Log10 (Base Pairs).
How do we best write the title of the graph?
1st write thing we are measuring
2nd the thing we know (Log10 base).
Do we plot the unknown distance with the known distance point on the graph?
No.
How do we plot the unknown distance on the graph?
Find the point –> draw a line across until meets the straight line –> draw line down to log10 base pair axis –> read off the value on x-axis.
What do we find with drawing the line down to the x-axis, of the unknown value?
The Log10 base pairs.
What do we need to find about the unknown distance value?
The base-pair size.
How can we find the base-pair size of the unknown distance value, with having its Log10 value?
Anti-Log of the value –> get value of base-pairs.
What s the result of the gel electrophoresis testing?
Finding the size of the band on an agarose DNA gel.
What did diabetics have to inject in their bodies many years ago?
Coe/Pig insulin.
What insulin do humans inject nowadays?
Human insulin.
From what is the human insulin produced?
Microorganisms: E. coli bacterium,
Certain yeast strains.
How do microorganisms produce human insulin?
With genetic engineering techniques.
What do scientists do in genetic engineering to turn microorganisms into human insulin for diabetics?
Turn certain microorganisms into –> mini factories –> make useful substances –> improve health, environment, economy.
What does organism’s DNA make?
its genes –> code for all proteins organism needs –> survive .
For what does each gene code in microorganisms?
For a different protein/part of a protein.
What is genetic engineering?
Manipulation/changing of organism’s DNA.
What does genetic engineering involve?
Removing a gene from one organism (donor) –> transferring to another organism (recipient).
What is the recipient?
The transgenic organism/genetically modified organism.
Which are the 2 basic purposes of genetic engineering?
- Require large volumes of protein to be made –> use transgenic microorganisms –> produce large volumes of specific protein: insulin, growth hormone, vaccines.
- Organism has gene from different organism introduced –> give advantage –> genetically modified organism/transgenic: gene manufactures toxic chemical in bacterium –> introduced in plant –> make –> toxic plant to insects: caterpillars.
How do we transfer genetic characteristics from one organism to another? (insulin)
Gene for human insulin is on chromosome 11 at position 15.5 –> insulin gene cut from chromosome 11 with restriction enzymes.
Bacteria have small circular strands of DNA (plasmids) in their cytoplasm.
–> Plasmid –> extracted from bacterium cell –> plasmid –> cut with same restriction enzymes cut insulin gene from human.
–> restriction enzymes –> leave sticky ends.
One of 2 DNA strands is longer than other.
Same restriction enzymes used = sticky ends on both DNA strands = complementary.
Sticky ends –> joined with complementary base pairing.
–> insulin gene –> joined to plasmid by sticky ends using ligase –> modified plasmid reinserted into bacterial cell.
How can we manufacture large amount of human insulin?
Insulin –> into bacterium –> large volumes of protein (insulin).
What is the modified bacterium?
Genetically modified/transgenic organism.
Why is the bacterium organism called modified/transgenic?
It contains some human DNA + own bacterial DNA.
How is the bacterial DNA called?
Recombinant DNA = bacterial DNA recombined with human DNA.
Where is the bacterial cell placed?
In a fermenter.
What does a fermenter allow?
Rapid asexual reproduction in ideal conditions.
Which are the ideal conditions in a fermenter for asexual reproduction?
Optimal temperature
pH
Lots of food
What happens to the offspring due to the asexual reproduction of bacteria in the fermenter?
Offspring = all clones of original transgenic bacterium.
What are the characteristics of the clones bacterium?
All have identical recombinant DNA.
How can the bacteria survive in the fermenter?
Make their own genes = normal bacterial proteins
And human insulin gene –> make human insulin.
What has the fermenter become in genetics engineering?
A culture of mini factories –> producing human insulin.
What happens to the insulin once is produced?
Extracted –> purified –> packaged –> injected n diabetic humans.
What DNA do we digest?
Double stranded DNA.
What happens if we digest a circular plasmid with one restriction enzyme site?
We linearize.
Get a single fragment.
Why do we get a linear fragment from one circular plasmid restriction enzyme?
No additional site, fragments are generated.
How does gel electrophoresis feel?
Small, jelly.
What does the word ‘electrophoresis’ mean?
Carried by electricity.
To what does ‘electrophoresis’ refer to in the lab?
Movement of molecules: DNA, RNA, protein –> mobilized by electric field through substance.
How many electrodes occur in electrophoresis?
2: negative, positive.
What doe happen when we turn the machine on?
Two electrodes create a difference in charge on the 2 sides of the gel –> electric field.
By what is electrophoresis separation of molecules affected?
Molecules’ size, charge.
What does a charge cause to the molecule?
Move through the gel.
Attracted to positively charged pole.
What is happening through the electrophoresis gel?
Positively charged electrodes –> towards negative pole and the opposite.
What do neutral molecules do?
Do not move at all.
What does DNA do?
Always negatively charged –> towards positive pole.
What does agarose gel have?
Irregular hole, pores.
Like a sponge.
How does the situation of the agarose gel show the size of the DNA fragments?
Moving through a complex substance –> shows how quickly different sizes move through.
How will the longest DNA pieces move through the gel?
Will find more holes –> difficult to move on the complex substance of gel.
What do we need as a first step in gel electrophoresis?
Make the agarose gel.
How can we make the agarose gel?
Dissolve the sugar in a liquid –> boil it –> cool down –> thicken in a mould (like making a jelly)
= agarose –> electrophoresis buffer –> boil –> add to gel casting tray –> add DNA stain.
How does agarose come?
As a powder/pre-weighed tablet.
Why do we add a DNA stain to the agarose gel when making it?
Make samples visualization easier after.
How do we know where to place DNA samples on the gel?
Add comb at end.
When do we remove the comb from the gel?
Once the gel cools and hardens.
How do the wells look in the gel?
Little pockets.
Where do we place the gel once it cools down?
In a gel electrophoresis chamber.
With what do we cover the gel?
With running buffer.
Why do we cover the gel with running cover?
To guide electricity.
With what do we mix the DNA sample before we add it in the gel?
With a loading die.
Why do we mix the DNA sample with loading die?
For the gel to sit in.
For DNA to sink to bottom when added to the well with pipette.
Easily track movement of molecules through gel.
How do we know that the DNA samples have been added good in the wells?
From the colour of the dye.
What are the characteristics of the dye?
Negatively charged,
Coloured.
What do we do once our samples are loaded?
Close lid.
Turn on power of machine.
For how long does gel electrophoresis run?
20 mins.
How can we visualise the DNA?
Turn on the safe blue light on the blue gel machine.
Use a dark hood.
Take pictures with phone.
Why shall we use a dark hood?
To see the bands in bright room.
How does DNA bands move?
In straight line.
What can we say about the DNA samples?
They run through a single lane.
How can the DNA bands be realised?
They are bright lines in each sample lane.
How many fragments is each band?
Billion different fragments.
What is the characteristic of each band in the same lane?
Same length, identical.
What can the speed of each band tell us about each DNA band?
How big they are in comparison to one another.
Which band is smaller?
The band that travelled longer distance in the same time.
What do we want to find from gel electrophoresis?
The actual length of the fragments of DNA we are separating.
How can we better assess the size of the DNA bands?
Put DNA ladder.
How does the ladder act?
As a ruler on the left, we compare our samples to.
Which fragment will move the furthest through the gel?
The 100bp fragment.
What does gel electrophoresis allow us to see?
If a piece of DNA is present in the sample or not.
What can we find using a gel electorphoresis?
An infection.
Individual identification by fingerprinting.
What process does gel electrophoresis do?
PCR.
What do we need to know before we make the agarose gel?
The % of the gel.
The volume of gel (how much we need to make).
What buffers can we use to mix with agarose?
TBE Buffer = Tris/Borate/EDTA buffer.
Why should we not swirl the flask with agarose and buffer once we mix them?
Because agarose will stick to the flask –> will not dissolved.
What should we do once we put the flask with agarose + buffer in the microwave?
Loosen the cap of the flask.
Why should we loose the cap of the flask in the microwave?
To not explode.
For how long should we put the agarose + buffer mixture in the microwave?
45 seconds.
Where should we put the flask once removed from the microwave?
Room temperature –> cool down.
Where do we pour the agarose mixture once it cools down?
In the gel electrophoresis machine.
What else do we put once we put the agarose mixture in the machine?
A comb.
Where should we transfer the gel after it is still with the wells appeared?
In the gel tank where there is buffer.
How should we pour DNA samples in the agarose gel?
Put tip of pipette in well –> slowly pour sample in well.
How is the DNA sample chracterised?
Heavier than water.
What do the sample do?
Sinks down to the well.
What should we put once all the samples are loaded?
The lid.
On what does the voltage we use in the gel electrophoresis machine depend?
On how long the gel is.
On what does the time we run the DNA samples in the gel electrophoresis machine depend?
On the % of jell we have.
On the voltage we run the gel at.
What is the relationship between voltage and time for DNA samples?
Higher voltage –> shorter time for DNA to run.
What is the relationship between % and time for DNA samples?
Lower % of gel –> shorter time for DNA to run.
What should we do once the samples stop running?
Unplug the machine.
Take off lid.
How can we tell if the samples have finished running?
Look at dye migrated.
How do the samples run in gel electrophoresis?
Horizontally.
Why should we dilute buffers and dyes with DNA samples efficiently?
To control DNA samples in the wells.
Why is it important to know how much of DNA samples will be loaded in the wells?
To make sure the wells will hold the amounts.
What will happen if we load more DNA sample into the well?
Spill into the next well –> contamination –> false results.
What will bubbles at the tip of pipette cause?
Sample spreading –> sample loss.
What will happen if we do not load the sample into the well appropriately?
Sample will spilled out of the well.
What will happen if we do not remove the tip of the pipette once we put the DNA sample in the well?
Will remove some of the sample back in the pipette –> not quantitative –> false results.
What does Gel electrophoresis use?
Electricity.
Why does gel electrophoresis use electricity?
To separate DNA fragments by their length.
How are DNA ladders called?
Standards.
Where can standards prepared?
In the lab.
Pre-made.
Why do we use standards in gel electrophoresis?
Better estimation of samples.
See if samples contain DNA.
What do samples and standards contain?
Billion copies of same fragment –> form visible bands.
What do bands in the same position, in 2 different lanes, contain?
Fragments of same length.
Where do shorter fragments occur in the gel?
Lower (moved faster).
Do we need to run standards in each gel in electrophoresis?
Yes.
Why do we need to run standards at each gel in electrophoresis?
Because measurements change and we need to estimate the lengths of different bands.
Where can we build the table for our standard curve of the DNA samples we run in gel electrophoresis?
Excel.
How can we build our plot for gel electrophoresis in excel?
Scatterplot –> trend line.
How can we make the analysis of the plot of gel electrophoresis easier?
Convert DNA sized to Log10 base pairs.
Of which lane in gel electrophoresis samples do we build the plot for?
The DNA ladder (standard).
Why do we only plot the DNA ladder samples in gel electorphoresis?
To plot the line of best fit of estimation samples and compare the unknown values –> find them in the plot.
What do we always know about our DNA ladder?
The base pairs.
How can we sue the known values of base pairs we know for the DNA adder?
Base pair numbers –> Log10 base pairs –> plot a graph –> Log10 on x axis –> measure distance of each band on DNA ladder –> plot distance (mm) on y axis –> plot graph –> find unknown log base pair of known distance of DNA sample we run –> anti-log of value –> find value of unknown base pairs of DNA sample we run.
