DNA Flashcards
1
Q
What is a gel?
A
A gel is a water-retaining polymer network
2
Q
Give 5 examples of some biological gels
A
- Cartilage
- vitreous humour in eyeball
- Cytoplasm
- Hydrogel in nuclear pores
- Polysaccharides in seaweed
3
Q
What does the ‘central dogma’ explain?
A
The flow of genetic information
4
Q
Give me some examples of what proteins are involved in?
A
- metabolism
- structure
- function
- organisation
- regulation
- development
5
Q
What did Friedrich Miescher discover in 1869? and what did he name it?
A
He found acid molecules which were rich in phosphate and isolated from cells in the puss.
He called it ‘nuclein’ (DNA)
6
Q
Whats the location of DNA in Eukaryotes and Prokaryotes?
A
Eukaryotes:
in the nucleus in chromosomes (complexes of proteins and DNA –> chromatin)
Prokaryotes:
Not membrane bound, circular, supercoiled-naked DNA (not bound in a protein coat)
7
Q
State some comparisons between prokaryotes and eukaryotes?
A
Prokaryotes
- little internal structure
- 2 um in diameter
- 70s ribosomes
Eukaryotes
- membrane bound organelles
- 20 um in diameter
- 80s ribosomes
8
Q
Does the genome size vary between species?
A
Yes
9
Q
Roughly how many base pairs does the human genome have?
A
3 billion base pairs in the human genome
10
Q
Do more chromosomes means a more complex organism?
A
No
11
Q
what’s the relationship between the number of chromosomes and the number of genes?
A
there is no relationship
12
Q
Label this chromosome
A
13
Q
What is the product of cell division in humans?
A
2 diploid cells, each with 23 pairs of chromosomes
14
Q
What is meant by the term ‘junk DNA’?
A
genomes of eukaryotes which contain DNA of unknown functions
15
Q
How many copies of each chromosomes does a human cell contains?
A
2 copies
16
Q
Whats means by the term Homologous chromosome?
A
1 paternal and 1 maternal chromosome, that contains the same gene at the same loci
17
Q
Whats the Karyotype?
A
A full set of chromosomes
18
Q
Whats an abnormal karyotype? What can these lead to?
A
Any chromosomes number that deviates from the normal set of chromosomes aka. Karyotypes.
This can lead to genetic disorders
19
Q
Name 6 genetic disorders
A
- Down syndrome
- Turner syndrome
- Klinefelter syndrome
- Leukaemia
- Cystic fibrosis
- Sickle-cell disease
20
Q
What is the reason that one may have down syndrome?
A
their cells have 3 copies of chromosomes 21 (Trisomy 21)
21
Q
In 90% of down syndrome cases, what parent does the extra chromosome tend to come from?
A
The mother
22
Q
Whats turner syndrome?
A
Women with turner syndrome only have 1 X chromosome (this is an example of monosomy: where only 1 parent contributes the X chromosome)
23
Q
In 75-80% of turner syndrome cases, what parent does the extra X chromosome come from?
A
the mother
24
Q
Whats Klinefelter syndrome?
A
Men with Klinefelter syndrome have 1Y and 2X chromosomes (trisomy)
25
With Klinefelter syndrome, what parent does the extra X chromosomes come from?
it can come from either parent
26
What are the genetic conditions of downs, turner and klinefelter mostly caused by? what is this?
**non-disjunction:** where a pair of chromosomes fails to seperate during formation of gametes
27
What chromosomes is **Leukaemia** linked to?
the **Philadelphia chromosome**
28
In Leukaemia, what leads to the formation of the **BCR-ABL oncogenic fusion gene?**
The translocation of material between chromosomes 9 and 22
29
What chromosome is the **CFTR** gene found on?
chromosome 7
30
Whats the most common mutation that causes CF? what happens in this mutation?
**∆F508**
this is the deletion of 3 nucleotides that reuslts in the loss of an amino acid
31
What's the role of a normal CFTR channel? What happens in a mutant CFTR channel?
Normal CFTR channel:
Moves Cl- to the outside of the cell
Mutant CFTR channel:
Doesn't move Cl- outside of the cell. Leads to sticky mucus build up on the outside of the cell
32
Whats does **sickle-cell disease** affect?
Haemoglobin
33
Whats different with the strucutre of the cells containing haemoblobin with the people who have sickle-cell disease?
They have atypical haemoglobin molecules
The RBC are **cresent shaped**
34
Tell me the strucutre of haemoglobin
* 2 alpha and 2 beta subunits
* contains a haem group with a Fe2+ group
* quaternary structure
35
Name the genes that code for alpha and beta globin proteins
HBB codes for beta-globin
HBA codes for alpha-globin
36
Tell me about the main mutation in base codes that leads to sickle cell disease
A is chnaged to T at position 17 in the beta chain
This changes the codon GAG (glutamic acid) to GTG (valine)
37
Tell me some symptoms of sickle-cell diease
* anaemia (low number of RBC)
* frequent infection
* episodes of pain
* painful swelling of hands and feet
* vision problems
38
In 1928, what did **Griffith** set out to find out?
Whether DNA was the genetic material in cells
39
What did Griffith undertake his experiments with and what did he discover?
Undertook his experiments with **Sterptococcus Pneumonia** and injected it into mice
Discovered that the **non-virulent strains become virulent** when mixed with **heat-killed virulent strains**
this **transformation was permanent** which proved that something from dead cells was being transformed in living cells
40
Whats does the following mean?
1. S strain (smooth strain)
2. R strain (rough strain)
**1. S strain:** has a polysaccharide coat which helps invade the immune system and _its hard to fight infection_
**2. R strain:** has no polysaccharide coat which makes it prone to being cleared by immune system. this _enables mice to get rid of bacteria_
41
Tell me the steps to Giffith's experiment and what principle he determined?
In 1944, what scientist then decided to discover the transforming principle?
1. Killed the S-strain with heat and mixed with R-strain
2. This made an S strain
3. This is because something transformed R--\> S strain (this proved the **transformation principle**)
4. All descendants of the transformed bacteria were also infectious
5. Griffith didn't know what component of the heat killed S-strain was responsible for the change. He called this the **transforming principle**
In 1944**, Avery** set out to find the transforming principle
42
Tell me the steps to Avery's experiment and how they detemined that the transforming principle was DNA
NOTE: there were LOTS of steps :(
1. **Broke open heat killed bacteria with a detergent** which released contents of cell
2. Used a **centrifige** to seperate cellular components
3. To ensure the extract contained the transforming principle, the extract was **mixed with a living R bacterium**
4. Mixture was **incubated** and plated in a **petri dish**
5. **Colonies of S bacteria** appeared on dish
6. This indicated that the **R bacteria has been transformed**
******This allows Avery to figure out that a component of the extract was responsible for the transformation**
Extract components: Polysaccharides, Proteins, DNA, RNA
7. They developed a method to isolate each component and test each one for its ability to transform the bacteria
8. First, the **extract was mixed with an enzyme called S3** (this removed the polysaccharides)
9. sample treated with live R bacteriumand incubated on petri dish
10. **S colonies grew**. Therefore transformaiton had occurs without **polysaccharides**, meaning that was **not the transforming principle**
11. Next, the extract was treated with **protease** (removed the proteins)
12. Sample mixed with live R bacterium and incubated in a petri dish
13. **S colonies grew, so proteins were not the transforming priniciple**
14. The remaining extract contains no Polysaccharides or proteins and only contained DNA and RNA
15. **RNase** was used to **break down the RNA** in the extract
16. A sample of extract containing only DNA, was mixed with live R bacterium and incubated in a petri dish
17. **S colonies grew meaning that RNA was not the transforming principle**
18. Nucleic acid mixture was treated with **DNase** which **removed DNA**
19. Sample containing only RNA was mixed with live R bacterium and was incubated in a petri dish
20. **No S colonies grew meaning that DNA must have been the transforming principle**
43
In 1952, what did **Hershey and chase** set out to find?
Whether it was the protiens of nucleic acids that were responsible for passing along genetic material
44
What did Hersehy and chase use for their experiments and why?
they used **bacteriophage's** as they have many advantages
45
Tell me the advantages of using Bacteriophages?
1. they reproduce quickly
2. made up of only nucleic acids and proteins (meaning they dont contain any lipids or carbohydrates)
3. have a protein coat on the outside and nucleic acids on the inside
46
Tell me the stages to the **reproduction of bacteriophages**?
1. attach to outside of host cell
2. inject their genetic material into the cell
3. protein coat of bacteriophage stays on outside of host cell whilst the genetic material goes into the host cell
4. Bacteriophages genetic information halts the host cells functions
5. host cell starts to produce copies of bacteriophage
6. offspring and parent are gentically identical
7. host cell become porous and releases all of the new bacteriophages out which allows them to infect even more cells via the same method
47
Hershey and chase split the bacteriophages into 2 groups. One testing the protein coats and one testing the nucleic acids.
What did they label (radioactively label) the protein coats and nucleic acids?
Protein coat was labelled with
**35S**
Nucleic acid was labelled with
**32P**
48
Tell me the steps to Hershey and Chases experiment
1. allowed the 2 groups of bacteriophage (T2) to **infect the host cell** (E.Coli)
**This way they would know what component (protiens or nucleic acids) were responsible for inheritance as it would produce radioactive offspring**
2. The bacteriophages injected their genetic material into the host cell
the genetic material from the **35S** would not produce radio acitve offspring as genetic material no labelles
the genetic material from **32P** would produce radio active offspring
3. They 'blended' the host cell to **remove the protein coats** from the outside of the host cells
4. The protein coats dissolved in the liquid portion of the experiment, which is called the **Supernatant**
5. the solution is **centrifuged** to seperate the mixture into a liquid portion, containing the protein coats, and a solid portion containing everything else. the Solid portion is called the **pellet**
6. this experiment showed that **nucleic acids are responsible for inheritance**
49
Tell me the strucure of DNA
DNA contains
* pentose sugar- deoxyribose
* phosphate group
* organic nitrogenous bases: arginine, cytosine, thymine and gyanine
50
What bases are classed as the **pyrimindines** and the **purines?** How many rings do they have in their strucure?
**Adenine** and **gunaine** are **purines,** they have a double ring strucutre
**Thymine** and **Cytosine** are **pyrimidines,** they have a single ring structure
51
Identify the nitrogenous bases...
52
On DNA, the bases are located on the inside of the helix and the phosphates are on the outside, what does this mean for the polarity of DNA?
The inside is **hydrophobic** and the outside is **hydrophiic**
53
What are the 2 major bonds in DNA and what are they between?
1. **Phosphodiester bond:** this is between the phosphate group of one nucleotide and the sugar residue of the next nucleiotide
**2. Beta N-linked glycosidic bond**: a beta-glycosidic bond is between two carbons with different sterochemistry and a glycosidic bond is between a carbohydrate and another molecule
54
What was **Chargaff's rule?**
His rule stated that DNA from ant call of any organism should have a **1:1 ratio of pyrimidine and pyrine bases and that A=T and C=G**
55
Why don't **bacteriophage** follow **Chargraff's rule**?
They have a single stranded DNA
56
What did **Roslaind Frankline** and **Maurice Wilkins** do and what did they discover about the strucure of DNA?
**_What they did:_**
* dehydrated DNA and surrounded it with water to look how the strucutre chnages with different levels of water availability
* obtained X-ray diffraction patterns on DNA fibres
**_What they discovered:_**
* **10 repeting units** per arm
* X shape produced shows a **helix strucure**
* gap at position number 4 suggest's that theres a slight offset and theres interference which shows the presence of the **major and minor groove of DNA**
* **0.34 nm between nucleotides** (3.4 Å)
NOTE: 1Å (angstrom)= 10-10 m
57
What did **James Watson** and **Francis Crick** discover?
* **Bases** were on the **inside**
* **phosphates** on **outside**
* between A and T there's **2 hydrogen bonds**
* between G and C there's **3 hydrogen bonds**
* **bases are hydrophobic**
* base pairing is **complementary**
* two strands are **anti-parellel**
* **right handed double helix**
* **base pairs are perpendicular to the helix axis**
* centre of **helix runs through base pairs**
* known as **B-DNA**
*
58
Tell me the key features of **B-DNA?**
1. right handed
2. 0.34 nm between base pairs
3. 3.4 nm per turn
4. 10 bp per turn
5. roughly 2 nm in distance
6. has 2 grooves of unequal size
59
Tell me all the possible combinations of DNA possible?
**A-DNA**
**Z-DNA**
**B-DNA**
60
Tell me the features of **A-DNA?**
* right handed
* formed when DNA is dehydrates
* wider
* bases tilter
* offset form axis
61
Tell me the features of **Z-DNA?**
* left handed
* formed by some GC-containin sequwnces at high salt concentrations
62
Identify which DNA conformation these are...
63
Whats the **hyperchomic effect** of DNA ?
This effect is the increase in **absorbance** of a material for instance DNA with UV light which leads to the denaturing of the DNA
64
What has a higher absorbance, single or double stranded DNA and why ?
**single stranded has a higher absorbance** as the bases and no longer stacked on each other
65
Whats a decrease in absorbance called?
**Hypochromicity**
66
do high salt concentrations give an increased or decrease in the melting temperature of DNA ? Why is this?
**A high salt concetration leads to a higher melting temperature of DNA**
This is because;
* negative ions neutralise the repulsive negative phosphate groups on the backbone
* the salt makes negatively charged phpshates and exisitng repulsion between phosphates leading to a higher DNA melting temperature
67
How is DNA packaged into the nucleus?
The DNA is condensed in the form of **chromatin** and is then packaged into the nucleus
68
What does chromatin contains and what forms does this come in, within the nucleus?
Chromatin contains 5 main proteins called **Histones** (histones wrap DNA around it and pack it into the nucleus).
The forms of histones are;
**H1**
**H2A**
**H2B**
**H3**
**H4**
69
What do the histone proteins contain and what do they react with and why?
The histones are very basic and contains lots of **positively charged amino acids** such as; arginine and lysine
These interact with the negatively charged DNA **(electrostatic attractions)** due to the positive and negative charge
70
What forms the structure of the histone core and what is it described as?
**two copies** of **H2A, H2B, H3 and H4** combine to form an **octameric,** disk-like structure known as the histone core
71
Whats a **nucleosome?**
When DNA is wrapped around a histone core (roughly 1.6 times)
72
How many base pairs are found in the linking region between histone cores?
around 20 base pairs
73
What can histone cores also help to regulate?
gene function
74
Whats the role of the **H1 histone?**
* **linker** histone
* contracts both proteins and DNA
* **changes conformation of DNA** as it leaves the nucleosome which helps to compact the DNA
75
What were the three proposed theories for DNA replication and what was the idea within them?
1. **Conservative:** maintains the double helix and theres one parent and one daughter
2. **Semi-conservative:** parent strand provides a template for the daughter strand
3. **D****ispersive:** 50/50 mix of parent and daughter in each strand, alternating within the strand
76
What was **Meselson** and **Stahl's** experiment?
They grew bacteria on heavy (non-radioactive) isotope of nitrogen (**15N**): old DNA
cells containing labelled DNA were then transferred to a medium containing the normal isotop of nitrogen (**14N**): new DNA
The DNA was then isolated after 1, 2 and 3 generations
77
What did each generation show in Meselson and Stahl's experiment?
How was the DNA seperated?
**At zero:** all DNA has 15N
**After generation 1:** all DNA density was between 14N and 15N
**After generation 2:** 50% has density of 14N and 50% is intermediate
**After generation 3:** 75% has density 14N and 25% is intermediate
The DNA was seperated by **density gradient centrifugation**
78
What happens in each of these centrifugations?
1. **Aside on centrifugation**
2. **sucrose density centrifugation**
3. **cesium chloride density gradient centrifugations**
**Aside on centrifugation:**
* the heavier particles sediment the fastest
* not an equilibrium- everything goes to the bottom of the tube
**Sucrose density centrifugation:**
* samples applies to the top of a tube that already contains a gradient of sucrose concentrations (more concentration at bottom)
* not equilibrium method- everything goes to bottom of tube
* gives better seperation of the fractions if stopped at right time
**Cesium chloride density gradeint centrifugation**
* samples are dispersed in a concentrated solution of cesium chloride
* this is an equilibrium method
* under high centrifugal force the cesium forms a concentration gradient with the ions (denser at bottom of the tube)
* seperates the samples in accordance to density, NOT mass of shape
* DNA 'floats' in this position that corresponds to its buoyancy density
79
Why is **semi-conservative** replication described as being **bi-directional?**
replication starts at a fixed point and then the replication occurs out from the origin
80
What is the **replication fork** formed from and what is it's resting structure?
The fork is formed from **helicases** and its resting strucutre has two branching 'prongs'
81
What direction is the synthesis in the replication fork?
in the 5' to 3' direction
82
Tell me what is required for DNA replication?
1. single stranded DNA template
2. extended from an existing strand (primer)
3. The primer to be made from RNA and made from the enzyme primase
83
In the lagging strand of DNA, when made in short fragments in the 5' --\> 3' directions, what are the fragments joined together by?
**Okazaki fragments**
84
What's the name of the enzyme in DNA that joins the short fragments together?
**DNA ligase**
85
State two important uses of DNA polymerase reactions?
1. DNA sequencing
2. DNA amplification (PCR)
86
What does **DNA sequencing** reactions contain?
All the usual **dNTPs**, plus a small amount of modified nucleotide triphosphate- **ddNTP** (dideoxynucleotide triphosphate)
87
in DNA sequencing what has a lower concentration: ddNTP or dNTP?
lower amount of ddNTP compared to dNTPs
88
If the DNA sequencing reactions contains dATP, dGTP, dTTP, dCTP and ddATP, what causes the synthesis to stop?
synthesis will stop everytime **ddATP** is incorporated
89
How are DNA fragments seperated and what gel's can be used?
DNA fragments are seperated via gel electrophesis.
the gels that can be used are;
1. **Agarose**
2. **Polyacrylamide**
90
What type of gel is **Agarose,** what is it used for, whats used to visualise the DNA and what colour does it fluoresce
**_Agarose_**
* a physical gel
* used for **seperating double stranded DNA** fragments
* horizontal submerged gel
* DNA is visulaised by added a dye called **ethidium** which fluoresces **orange**
91
What type of gel is **Polyacrylamide,** what is it mainly used for, what is the DNA visualised by?
**_Polyacrylamide_**
* a chemical gel
* used for denaturing gels containing 8M urea, to **seperatre single-stranded DNA** fragments
* DNA is visualised by **autoradiography (32P)** or by **covalernt attachment** of a fluorescent group
92
What's **next generation sequencing?**
* **Short fragments of DNA** as input, annealed to a slide using oligonucleotide adaptors
* use of **PCR** to create multiple copies
* use of **fluorescent nucleotides** for sequencing
* use of a **reversable terminator**
93
What are the steps to **PCR**?
**1. denature** the DNA to produce single strands using heat
**2. add short primers** that are complementary to the ends of the sequence of interest
Lower temperature --\> anneal
**3. Termostable DNA polymerase**
* isolated from Termus aquaticus (Taq) as it is heat stable so wont denature (however has a high error rate)
* **require for DNA extension**
**4. repeat steps of denaturation, annealing and extension**
