7.1 DNA structure and replication (HL) Flashcards
Why did the Hershey-Chase experiment come about?
Scientists weren’t sure whether protein or DNA was genetic material
Why were viruses used in Hershey-Chase?
It was known that they could transfer genetic material to hosts so that host (bacteria) can replicate the virus. They also had DNA and a protein coat.
Hershey-Chase: Why was sulphur used in one while phosphorus in the other?
Used to label and distinguish between protein and DNA. Proteins have sulfur but no phosphorus while DNA has phosphorus but no sulfur
Hershey-Chase: Which viruses were used?
T2 bacteriophages
Hershey-Chase: Where were the viruses grown?
They were grown in one of two isotopic mediums to label component radioactively
Hershey-Chase: Which isotopic mediums were used?
S-35 and P-32
Hershey-Chase: What happened after viruses were labelled and what was the separation process called?
They were allowed to infect E. coli and then the virus and bacteria were separated through centrifugation
Hershey-Chase: What is a supernatant?
It is the liquid lying above the residue after precipitation or centrifugation
Hershey-Chase: What was the result of centrifugation and why was it done?
Larger bacteria formed a pellet (sedimentation) because it was heavier while viruses remained in the supernatant. This means genetic material should be in pellet while non-genetic material remains in supernatant.
Hershey-Chase: Which isotope was found in pellet and which in supernatant? What does it show?
- In P-32 condition, the pellet was radioactive
- In S-35 condition, the supernatant was radioactive
- This showed that DNA was genetic material as viruses only transfer genetic material while proteins were not
Franklin-Wilkins: What do X-rays do when they pass through a substance?
They diffract and scatter
Franklin-Wilkins: What should the material be for X-ray diffraction to work well and why?
They should ideally be crystallized so that repeating pattern causes diffraction to occur in a regular way
Franklin-Wilkins: What could be deduced from X-shaped pattern?
Shows that DNA was a helix
Franklin-Wilkins: What can be deduced from regular nature of pattern?
Dimensions of helix were consistent eg. diameter of helix and distance between strands
Franklin-Wilkins: What does vertical distance between horizontal bars show and what was the measurement?
Shows distance between two stacked base pairs. Each distance was 3.4 angstroms
Franklin-Wilkins: What does the distance from middle of image to top measure show in DNA?
Height of a helical turn (34 angstroms)
Franklin-Wilkins: How can you deduce number of bases in a helical turn?
Height of turn/ Distance between horizontal bars. i.e. 34/3.4= 10 base pairs per turn
Franklin-Wilkins: What can be deduced from angle between horizontal axis and arms of X-shaped pattern?
The helix’s pitch or degree of its rise
Franklin-Wilkins: From the images, what deduction did Franklin make about positions of molecular units within helical structure?
She deduced that phosphate groups were on the outside
What mechanism aids DNA replication?
Complementary base pairing
Why can C only pair with G and A only with T?
Electrical charges of adenine and thymine are compatible as they are opposing (2 H bonds). Same for cytosine and guanine (3 H bonds)
Why is prokaryotic DNA considered naked?
Although DNA is supercoiled, they don’t have histones to be wrapped around like eukaryotes
What are histones?
Proteins used by cell to package DNA
What does a nucleosome consist of?
- Central core of 8 histones (octomer)
- Core DNA+ short linker DNA
- Additional histone called H1 which binds DNA to core histones for further packing
What are nucleosomes bunched together usually known as?
30nm fibers or solenoid
What is supercoiling?
When a DNA strand has been wound back on itself so molecule becomes compact
How many times is DNA wound around histone?
Twice
Charges of DNA and histones
DNA is negatively charged and acidic while histones are positively charged and basic
Why is it essential to supercoil chromosomes?
- Packs genetic material into tiny nucleus
- Allows chromosomes to be mobile in mitosis and meiosis
- DNA cannot be transcribed for protein synthesis (controls expression)
- Allows cells to specialize by permanently supercoiling DNA that’s not required
Which part of cell cycle is DNA most supercoiled in?
Meiosis and mitosis
Which part of cell cycle is DNA most supercoiled in?
Meiosis and mitosis
What is a primer
They are short stranded sections of DNA/RNA.
Difference between RNA and DNA primers
RNA primers initiate activity of DNA polymerase while DNA primers are used in PCR
Which direction does DNA polymerase move along template strand?
5’ to 3’ direction
What is difference between lead and lagging strand?
Replication is continuous on lead strand and discontinuous on lagging due to anti-parallel nature. Can only move from 3’ to 5’ hence why primers are required on lagging strand and creates fragments
DNA helicase
Unwinds and separates DNA by breaking H bonds between bases. Helps create the fork of two strands
DNA gyrase/ topoisomerase
Reduces strain created by unwinding of DNA by helicase. (Extra: Uses -ve supercoiling to relax +ve supercoils)
DNA primase
Generates short RNA primer on template to provide initiation point for DNA polymerase III.
DNA polymerase III
Forms covalent bonds between nucleotides as they are added to polynucleotide
DNA polymerase I
Lagging strand has multiple RNA primers so they remove them and replace with DNA nucleotides
DNA ligase
Joins Okazaki fragments to form continuous strands. Covalently joins sugar and phosphates with phosphodiester bond.
SSB proteins
Stands for Single Stranded Binding proteins. They prevent template strands from reconnecting so they can be replicated
Okazaki fragments
They are the RNA primers + DNA fragments
Difference between coding and non-coding regions of DNA
- Coding regions contain sequences for polypeptide production
- Non-coding regions make up most of genome and help regulate gene expression.
How do non-coding regions regulate gene expression?
They act as enhancers or inhibitors for genes which affects the genes that get transcribed to mRNA
Which regions of DNA have highly repetitive sequences and what is their function?
Telomeres. They are the end of chromosomes and prevent degradation of DNA during replication
What was known as ‘junk DNA’ and why?
Non-coding regions as they were thought to have no function
Non-coding regions: Introns
They are within the gene and removed by splicing prior to formation of mRNA They do not contribute to polypeptide production
Non-coding regions: Gene regulatory sequences
Sequences involved in the process of transcription. Includes promoters, enhancers and silencers
Non-coding regions: Non-coding RNA genes
They code for RNA molecules that do not get translated eg. forming tRNA molecules
How does dideoxyribonucleic acid affect DNA replication?
DNA polymerase requires a nucleotide with a 3’ OH group to form bond however ddNTP only has 3’ H group so it stops replication
What does ddNTP stand for?
Dideoxyribonucleic triphosphates
What is attached to ddNTP during base sequencing?
Fluorescent markers so base present when replication stops is identified
What is it known as when DNA synthesis stops due to ddNTP?
Chain termination method or dideoxy DNA sequencing
Sanger sequencing: How many reactions take place and what do they consist of?
4 reactions occur parallelly. They contain copies of DNA template strand, radiolabeled primers, DNA polymerase and all forms of dNTP (dATP, dCTP, dGTP and dTTP). One type of ddNTP will be added to each
Sanger sequencing: How are multiple DNA molecules generated?
PCR
Sanger sequencing: If sequence is TACGGATGATC and ddATP is added, what will all fragments look like?
- 5’ A 3’
- 5’ ATGCCTA 3’
- 5’ ATGCCTACTA 3’
Hence we know T is first, seventh and tenth in sequence
Sanger sequencing: What are the contents of each reaction loaded into?
DNA electrophoresis gel
Sanger sequencing: How do contents separate in electrophoresis gel?
They separate on basis of their length. Smallest fragments travel down the most.
Sanger sequencing: What is autoradiography?
It is a photograph of gel produced by radiation. It labels lanes according to dNTP with which each fragment should end.
Sanger sequencing: How do you read sequence in the gel?
Read it from bottom to top. This is newly synthesized strand. Old template strand will be complementary to the fragments in the gel
What is VNTR?
Variable number tandem repeats. They are short nucleotide sequences that show variations between individuals in terms of number of times sequences is repeated consecutively.
Where is VNTR used?
They can be used for genealogical investigations using allele combinations
What is a locus?
Physical location of heritable element on chromosome
2 different sources of DNA used in parental profiling
- Paternal lineage: Short tandem repeats from Y chromosome
- Maternal lineage: Mitochondrial DNA variations in single nucleotides in hyper-variable regions
Why are non-coding regions more useful in DNA profiling?
They are used for gene expression and so we can compare differences in alleles
Why are tandem repeats used in DNA profiling?
Everyone has different tandem repeats so they can be combined to map a DNA profile and match individuals.
How many tandem repeats in GTCTACTACTACTATGGT?
CTA is repeated 4 times
What should you look for when given a question on matching DNA profiles (like parentage)?
Look at which profile doesn’t have a lone allele as child will inherit different VNTRs from both parents
