08/14/18

1) Question: What is the study of genetics?

Answer:​ In essence, the study of heritable elements (genes)...refer to information

passed from parent to offspring.

2) ​Question: What areas are present within genetics

Answer:

● Transmission Genetics - mechanisms and rules by which genes pass

down
● Molecular - look at molecular level
● Population - genetic variation within populations and changes over time

3) ​Question: How can you approach genetics? [two primary approaches]

Answer:
● Forward genetics (phenotype -> genotype)
○ Natural or artificial phenotypic variants
○ Show that it has genetic basis and underlying variation
○ Identify molecular basis (through sequencing)
● Reverse genetics (genotype -> phenotype)
○ Identify gene of interest and mutate it to identify its effect upon
phenotype
○ EXAMPLE: Finding gene for cystic fibrosis within a mouse and
mutating it to see what occurs

Question: What was griffith’s experiment?

Answer:
1. He injected mice with virulent (S strain) streptococcus pneumonia and the mice
died
2. If injected with nonvirulent bacteria (R strain), they didn’t die
3. If heat-killed virulent bacteria injected, they lived
4. If heat-killed virulent bacteria mixed with live nonvirulent bacteria, the mouse died
Conclusion: ​A substance was transferred from the heat-killed virulent bacteria to the
nonvirulent bacteria, transforming them into virulent bacteria...this substance is the
heritable unit

Question: What was avery’s experiment?

Answer: ​They discovered the chemical nature of the transforming substance. They
found that the R-type bacteria was transformed into S-strain bacteria always unless the
DNA was destroyed. This helped confirm that DNA was the source of heritable genes.

Question: What was the hershey chase experiment?

Answer: Ejected proteins with radioactive sulfur to see if it was transferred to another
bacteria. Then, did the same thing with radioactive phosphorus. The phosphorus was
transformed, meaning DNA was the heritable substance since phosphorus is only
present within DNA while sulfur is only present within proteins. Bacteriophages were
used to inject these substances into the bacteria.

**ADENINE AND GUANINE ARE DOUBLE RINGS (PURINE)

** CYTOSINE AND THYMINE ARE SINGLE RINGS (PYRIDINE)

CHARGAFF’S RULE: ​Adenine = Thymine and Cytosine = Guanine

Question: ​What did rosalind franklin’s data indicate?

- Simple structure, long and thin

- Probably two strands, parallel and helical
- Phosphate on the outsides (not inside)
08/16/18

Question: How was the structure of DNA elucidated?

● Linus Pauling
○ Solved structure of collagen and proposed triple helix for DNA with bases
on the outside
○ Didn’t have X-ray diffraction data
● James Watson, Francis Crick
○ Had access to Franklin’s diffraction data
○ Proposed a double helix model
● The C-G base pair has three bonds and is a 50% stronger interaction than the
A-T base pair which has two bonds
○ Phosphate groups on the outside using covalent bonding and use
hydrogen bonding to bond base pairs

QUESTION: IMPLICATIONS OF DNA STRUCTURE?

● Blunt double cut -- cut on both sides of the molecule at adjacent bonds to create
two single-strands??
● Staggered double strand cut - cuts at two places but not adjacently and will leave
an overhand (leave staggered ends)
● Single strand cut (nick) - only cuts in one place...leaves molecule intact but gives
it a greater ability to wiggle and can be reattached
● Two strands can be separated (denatured) without breaking covalent bond
○ As GC content rises, DNA is harder to denature (3 bonds vs. 2)
● Molecule is long (nucleus is ~ 10um)
● Information in linear sequence
○ Unlimited number of possible sequence
● Obvious means of copying information
○ Either strand can be used to obtain information for entire helix
● ***Single stranded DNA absorbs light at 260 nm at a much better rate than
double-stranded DNA

CONCLUSION: ​It was interesting that as soon as Watson/Crick released their paper,
everyone knew it was right due to the simplicity. Amazing how DNA is tightly wound into
a cell
QUESTION: How does dna replication occur?

● Semiconservative replication was the most likely choice

○ Half of parent strand is preserved within the replicated DNA
○ Meselson and Stahl experiment: USED DENSITY GRADIENTS
■ Grow cells in 15N for multiple generations and then in one
generation of 14N and then a second generation in 14N
■ Allows different densities to be separated out
● Occurs bidirectionally from origins of replication in E. Coli {more complicated in
humans}
○ There are thousands of these replication sites in eukaryotes
● Replication bubble with two replication forks from each origin are created
● DNA POLYMERASES
○ Synthesise 5’ -> 3’, read 3’ -> 5’
■ Two strands are replicated in opposite directions at a replication
fork
■ One strand discontinuous replication (lagging) and one continuous
(leading) strand
○ Cannot initiate replication of ssDNA; need a primer
■ Multiple priming on one strand
■ Chromosome ends are problematic (have to use telomeres)
■ The new piece of DNA is antiparallel, meaning it will be read in the
opposite direction
○ 3’->5’ exonuclease activity is used for error removal
■ Error rate is 1 error per 10^5 base pairs
○ Some have 5’->3’ exonuclease activity {very important because you can’t
replicate without them}
○ Termination of replication
■ Polymerases continue until they run out of template, or reach 5’
phosphate of double-stranded region (if no 5’-> 3’ exonuclease)
■ We lose small regions off end of each generation so after 30-50 cell
divisions, apoptosis is triggered
08/21/18

Question: What is ​PCR

○ Method for amplifying single region of DNA up to ~10kb {has to be less
than 10,000 base pairs} (developed by Kary Mullis)
○ EXTREMELY SENSITIVE (1 molecule of DNA enough for template)
○ Requires knowledge of the region’s sequence
■ For 2 ssDNA primers (18-30 bases) and uses a heat resistant DNA
polymerase (Taq polymerase)
■ Done in a thermocycler which uses high heat
■ STEPS IN PROCESS:
● 1) Heated to 90-100 degrees Celsius to separate two
strands
● 2) DNA cooled and single-strand primers allowed to anneal
to their complementary sequence
● Solution is heated to 72 degrees Celsius and DNA
polymerase synthesizes new DNA strands, creating two new
double-stranded DNA molecules
○ As cycle is repeated, the amount of target DNA in
existence doubles
● ***In the last cycle, you don’t do denaturation, so everything
will be double-stranded. If you heat it up, you can get
single-stranded
○ Has changed molecular biology BY CREATING unlimited amounts of
targeted DNA
○ Changed diagnostics for cancers
■ Extremely sensitive screening for known translocation
○ Changed forensics
■ Can amplify short amounts of DNA and help with fingerprints
■ Inclusionary
● Can be used to prove a crime vs. blood type which can only
prove that someone didn’t do it

Conclusion: ​ PCR is a powerful technique that can be used to amplify small sequences
of DNA. High heat is integral to the process along with DNA polymerase and primers.
Question: What are microsatellites?

● Variables number of tandem repeats (VNTR)

○ 2-20bp per repeat (ex: AGAGAGAGAGAGAG)
○ Don’t encode protein but tend to be in random part of DNA
● These evolve rather rapidly with 10-30 alleles per locus
● Forensics
○ Probability of an exact match: 1 locus: < 0.02 while for 10 loci: < 10​-17
○ Thus, can easily confirm perpetrators
○ Ex: If you find blood, you can genotype it and find that on the pair of
chromosome 3, there are 12 and 14 repeating units. Thus, only someone
with the same number of repeating units could be a suspect
■ ****Read more into the chart of microsat locus

Answer: ​Allows us to analyze meaningless parts of DNA to give us an increased

chance of matching DNA found in one area to suspects

Question: Why is DNA sequencing important?

● Sanger dideoxy sequencing

● Requires ssDNA template and primer
● Four reactions
○ All four dNTPs in each reaction
○ One of four ddNTPs per reaction
■ THE ddNTP does not have a 3’ hydroxyl group which prevents
additional polymerization and stops the reaction
■ ***look at video of how this works---??? Kind of confusing
● Reaction stops when ddNTP is incorporated
● The Sanger Capillary Sequencing process adds on to this process by giving each
DdNTP (ddTTP, ddCTP, etc.) a different color to represent each base...laser
detection of colors gives us a chromatogram
○ Understand diagram of bridge amplification
● SEQUENCING BY SYNTHESIS
○ The DNA polymerase will add one base and then stop (every base is like
a ddNTP) → but reason for stoppage is because they have a blocker
○ After each stopping, dye present is scored and then block is removed so
next base can add
08/23/18

Question: ​What is the central dogma of molecular biology?

● DNA can be transcribed into RNA, which is then translated into protein. The
reverse of RNA -> DNA has also been shown (Ex: telomerase)...replication also
occurs within these steps {check out chart on powerpoint}

Question: ​Function of RNA

● Found in nucleus and cytoplasm

○ Some viruses lack DNA (single-stranded RNA is only present)
● Obvious means of encoding and amplifying DNA information
○ When transcribing DNA into RNA, you can amplify it
● Composed of ribonucleotides (vs. deoxyribonucleotides in DNA)
● More reactive than DNA and usually single stranded and can form short
stretches of double stranded RNA-RNA or RNA-DNA
● Synthesized by RNA polymerases
○ (termed transcription if template is present)
○ No primer is needed and synthesized 5’ -> 3’
○ Read 3’ -> 5’
○ Error rate is 10​-5​ mis-incorporations per base (IMP: Can make a mistake
and immediately remove it but if next nucleotide is added, can’t go back
and fix it unlike DNA polymerases)
● Types of RNA
○ Messenger RNA (mRNA)
■ Short-lived {seconds to hours} and transcribed from protein-coding
genes
■ Often processed before translation
○ Transfer RNA
■ Used during translation
■ Deliver amino acid to growing polypeptide chain
■ Many modified bases
■ ***Anticodon is the part that interacts with the mRNA
■ ***3’OH is the attachment site for amino acids {the CCA bases near
this hydroxyl is added post-transcription {known as processing}
■ Lots of modifications along the way (like thymine added in or
inosine or pseudouridine)
● ***Also have non-watson-crick base-pairing
 ○ Ribosomal RNA
■ Present in ribosomes (most common type)
○ Small nuclear RNA (snRNA)
■ Play a role in mRNA processing
○ Micro RNA (miRNA)
■ Posttranscriptional regulation of mRNA
○ Small interfering RNA (siRNA)
■ Involved in viral and transposon defense
○ Many other types also present

Answer: ​Has many functions and is often single-stranded. Not the molecule of
inheritance so errors are more likely. Has many subtypes which each have their own
function

Question: ​How does transcription occur?

1. RNA polymerase core enzyme bind to -10 and -35 promoter sequences
2. DNA unwinds near the start of transcription to form the open promoter complex
(now you have single-stranded DNA)
3. Termination can be rho-dependent
a. Requires binding site for rho protein (rut site)
b. Rut site is transcribed from a sequence in the DNA and ends mRNA by
pulling it out of the DNA sequence
4. Termination can be rho-independent
a. Requires formation of stem loop in newly synthesized mRNA

Question: ​How does transcription differ in eukaryotes?

● Initiation involves interaction between RNA polymerase complex and core and
regulatory promoters
○ Proteins involved in DNA packaging affect transposition
● Core promoter sequences are important
○ CAAT box, TATA box, etc.
● Enhancer and silencer elements important
○ Bind transcription factors that interact with promoter-bound proteins
● Different classes of genes transcribed by different polymerases (DNA I, II, III)
 ● TRANSCRIPTION
○ Could be in the absence of protein binding (involves stem loop
sometimes)
○ Could be following protein binding at DNA sit (rut site)
○ Some termination following RNA cutting by nuclease at poly A signal
● Newly transcribed mRNA must be processed from pre-mRNA to mature mRNA
○ 5’ capping (7 methyl guanosine)
■ Added ‘backwards’ by capping enzymes (protects nucleic acid from
exonucleases which degrade the ends)
■ Enhanced stability and recognition
○ 3’ polyadenylation
■ Added by template-independent RNA polymerase following signal
sequence in RNA
■ 20 to 200 adenine ribonucleotides added
■ Enhanced stability and recognition
■ Done by a template-independent RNA polymerase
○ Intron splicing
■ Remove non-coding regions from mRNA
■ Alternate splicing possible
● Once processed, it leaves the nucleus

08/28/18

Question: ​What are the major questions surrounding translation?

● Do the codes overlap?

○ They found that there is no overlapping because a genetic mutation in one
base would change three consecutive codons…
○ HOWEVER, in nonoverlapping, one codon would be changed with one
base pair mutation
■ Bacteriophage T4 was used to determine how long codon
sequences were
● You grow them on the bacteria and they lyse the bacteria
and leave spots (small spots created by normal vs. big spots
created by mutant bacteriophages)
○ HOWEVER, mutant can only infect E. coli B strain but
not E. coli K strain
 ● Isolated numerous frameshift mutations (suggests fixed
starts)
○ Essentially no reversion to normal type unless
proflavin is added
● If you make a mutant with three insertions or three
subtractions, you go back into the reading frame
● AUG (methionine) -> is the start codon {but note very AUG is a start codon}
● UGA, UAG, UAA are the stop codons
○ Code is degenerate but not random (most of degeneracy is at 3rd
position) -> leads to wobble {....U/C are interchangeable in the third
position since it always leads to production of the same amino acid/
almost the same for A/G}