D1.1: DNA Replicaiton Flashcards
(26 cards)
What is DNA replication?
The production of exact copies of DNA with identical base sequences
- Semi coservative
- Depends on the complementary base pairing
- takes place in the nucleus
Important in multicellular organisms for growth, replacement of damaged/old cells, reproduction
- mitosis generally (DNA doubled before parent cell can divide -> 2 identical daughter cells)
What does semi-conservative mean in the context of DNA replication? Why is it so important?
In each new DNA molecules produced:
One strand of the DNA is from the original DNA
One strand of the DNA is from the original DNA (template strand) being copied
Important because:
Ensures genetic continuity and high accuracy
- ensure new cells produced inherit genes with correct DNA base sequences
What are the different bases in DNA? Why is complementary base paring so important?
Adenine + thymine, Cytosine + guanine
Ex: if adenine next exposed base on template strand, thymine added
Added one by one to the new strand based on complementary base paring
- hydrogen bonds can only form between template and new strand if correct bases are paired
= DNA is half parent, half new
What does DNA polymerase do?
DNA polymerase -> enzyme
Links nucleotides (covalent bonds) together to form a new strand, using the pre-existing strand as a template
Explain Crick and Watson explanation for semi-conservative replication
While discovering double-helix structure of DNA -> made hypothesis about how DNA copies during cell growth
Structure = evidence for complementary base paring -> key for replication
Proposed a semi-conservative model -> BUT NO EVIDENCE
Evidence can later with 2 other scientist: Meselson and Stahl
-> analysis of their research gave evidence needed to support C&W hypothesis
- proposed the DNA ‘unzipped’ -> H bonds broken between bases
- new strand then synthesized using original as template
What does DNA helicase do?
DNA helicase -> enzyme
Unwinds DNA at the replication fork - flatten helix structure
Causes H bonds to break between base pairs -> bases exposed
Each polynucleotide DNA strand -> template for formation of new strand using free nucleotides (attracted to exposed DNA via base pairing)
After DNA helicase unzips DNA, what does the DNA polymerase do?
After helicase -> template strand exposed + new nucleotides joined together by DNA polymerase
- catalyze condensation reaction
DNA polymerases ALWAYS build DNA strand in 5’ to 3’ direction
- attach to 3’ end of template strand and read strand 3’ to 5’
- DNA strands antiparallel -> new strand is being built in 5’ to 3’ direction
DNA polymerase help form H bond between complementary base pairs of the template and the new strand
What is gel electrophoresis?
A technique used in the analysis of DNA, RNA and proteins
Molecules are separated using an electric current according to their size/mass and their overall charge
How does gel electrophoresis separate molecules based on charge and size?
Electrical charge:
Positively charge molecules move to cathode (neg)
Negatively charged molecules move to anode (pos)
Ex: DNA neg charge (phosphate group) -> electric current -> molecules move towards anode
Size:
Different size of molecule -> move through gel (agrose of DNA, polyacrylamide for protein) at different rate
Tiny pores in gel -> small molecules quick, larger molecules slow
- different types of gel -> different sized pores affect speed
Outline how gel electrophoresis is carried out to separate DNA fragments
- Make agrose gel plate in a tank -> create wells (rectangular holes) in one end of the gel
- Submerge gel in an electrolyte solution (salt solution) in the tank
- Load DNA fragments into the wells using a micropipette
- Apply an electrical current to the tank -> neg electrode (cathode) on end with DNA, pos electrode (anode) on the other side
- DNA fragments move to anode due to attraction and moves away from cathode because of repulsion - DNA fragments will small mass -> faster than larger -> move further
- Fragments aren’t visible to the naked eye -> transferred onto absorbant paper or nitrocellulose -> heated to separate the two DNA strands
- Probes added to develop visual output. Either:
A) radioactive label: cause probes to emit radiation that make X-ray film go dark -> dark bands
B) fluorescent stain/dye: fluoresces when exposed to UV -> colored bands
What is PCR?
Kary B Mullis -> conceptualized 1983
Polymerase chain reaction: in vitro method of DNA amplification
- used to produce large quantities (billions) of specific, identical fragments/copies of DNA/RNA from very small quantities
Done in a few hours
Each cycle -> amount doubled
Allow enough DNA/RNA for analysis
Used in molecular biology, DNA profiling, genetic engineering
What are the requirements of PCR?
Target DNA/RNA that is being amplified/primer:
- whole genome doesn’t need to be copied - only specific sections that we know are different between individuals
- identified by adding a primer sequence that binds to them
DNA polymerase:
- enzyme used to build new DNA/RNA
- Most commonly used Taq polymerase (thermophilic bacterium Thermus aquaticus)
-> used because it doesn’t denature at high temps (needed in PCR)
Free nucleotides:
Used in the construction of DNA/RNA copies
Buffer solution:
Provide optimum pH for reaction
What are the main stages of PCR?
In each cycle DNA doubled -> standard is around 30 cycles -> million DNA molecules
Required special equipment -> thermal cycler (automatically provides optimal temp for each stage + time)
- Denaturation: double stranded DNA heated to 95°C -> break H bonds between base pairs
- Annealing: the temperature decreased to 50-60°C so primers can anneal to the ends of the single strands of DNA
- Elongation/extension: the temperature increased to 72°C for at least 1 min (optimum temp for taq polymerase) -> build complementary strands of DNA -> produced identical double-stranded DNA molecules
Why is PCR used?
DNA profiling -> allow individuals genes to be visualized
Allow someone’s genetic makeup to be compared to known genes -> see if they have
Used to identify genetic disorders/match samples
-> usually introns are sampled -> unique to individuals (not twins tho)
-> repetitive regions = STRs (short tandem repeats)
How can we get STRs?
- DNA amplified using PCR -> make primer match target sequence
- Restriction endonuclease (restriction enzymes) cut DNA where a certain DNA sequences appears -> fragments
- Fluorescent marker bind to a triplet in the DNA fragment -> results seen
- Sample added to gel electrophoresis -> separated by size using electric field
How is PCR and gel electrophoresis used in DNA profiling?
DNA profiling/genetic fingerprinting -> allow identification of suspects of a crime and identify corpses
Everyone (besides twins) have short, non-coding regions of DNA (20-50 bases long) that are unique
- VNTRs (variable number tandem repeats)
Gel electrophoresis: separate VNTRs fragments according to length -> create a pattern of band that is unique to every individual
How is a DNA profile created?
- Obtain DNA (extracted from root of hair, blood, semen, saliva)
- Increase the quantity of DNA using PCR
- Use restriction enzymes to cut the amplified DNA molecules into small fragments
- Separate the fragments using gel electrophoresis
- Add radioactive or fluorescent probes that are complementary and therefore bind to a specific DNA sequence
- X-ray/UV used to produce images of the radioactive/fluorescent labels
- Images contain patterns of bars -> analyzed and compared
How is DNA profiling used in paternity investigations?
Every persons -> inherits VNTRs from mother and father
DNA profile of mother and child compared along with profiled of potential father
- pattern of bands compared on all three profiles
Any band that appears in the child’s profile MUST show in either the father or mothers profiles -> if not alleged father is not the father
How is DNA profiling used in forensic investigations?
Can be used to identify suspects of crimes or identify bodies/body parts that are unidentifiable
Can also eliminate innocent people
Samples of body fluid are taken from crime scene or victimes body
DNA removed and profiled
Profile compared to samples from suspects, victim and people with no connection to the crime (control)
Must avoid contamination!
Explain how bases bond
5’ end -> phosphate group attached to 5’ carbon on deoxyribose sugar
3’ end -> -OH group attached to 3’ carbon on deoxyribose sugar
DNA replication -> must occur in 5’ to 3’ direction
- add nucleotides to the 3’ end of the template strand
when polymer made adds new nucleotide -> 5’ phosphate group of INCOMING DNA nucleotide bonds with the free 3’ -OH group of the GROWING STRAND
What is the leading and lagging strands?
Double-stranded DNA -> two antiparallel strand
BUT DNA polymerase can only add nucleotides in the 5’ to 3’ direction
-> replication needs to happen in OPPOSITE DIRECTIONS
= each strand is synthesized differently
Leading: made continuously after the fork as it opens
Lagging: made discontinuously in short fragments (called Okazaki fragments) away from the fork
- later joined together by DNA ligase
What are SSB proteins?
Single stranded binding proteins:
Keep the separated strand apart whilst the template strand is being copied
What is DNA primase?
Generates a short RNA primer on the template stand -> provide initiation point for DNA polymerase III to add new nucleotides
What are the 2 types of DNA polymerase involved and what do they do? (+ DNA ligase)
DNA polymerase III:
Starts replication next to RNA primer linking nucleotides in the 5’ to 3’ direction to form a new strand
DNA polymerase I:
Removes the RNA primer on the leading and lagging strands and replaces them with DNA
DNA ligase:
Joins the Okazaki fragments by catalyzing the formation of sugar phosphate bonds -> backbone
