Lecture 2+3

Amino Acids make which macromolecule?

proteins

fatty acids make which molecule

phospholipids

Carbohydrates make .. (1)

glycerol

Carbohydrates make .. (2)

Monosaccharides

glycerol makes which molecule

phospholipids

Monosaccharides make which molecule

Polysaccharides

nucleobases make which molecule

nucleotides

nucleotides make which molecule

DNA/RNA

What are the 3 components of a nucleotide

phosphate, sugar, base

what is the difference between a nucleoside and a nucleotide

a nucleoside has no phosphate

what charge does dna have

negative

what is the structure of DNA

double helix

which ends are attracted to each other in the DNA structure

5'-3'

how is the individual strand held together

the backbone is held by covalently bonded phosphates which share electrons and are hard to break

how are the two strands held together

the two strands are held together by hydrogen bonding: sticky, not permanent, can separate, weaker

in DNA A ->

T

C ->

G

what happens if you pull on both strands of a molecule of dsDNA

it behaves like a spring

what is a somatic cell

a cell that has two copies of each genome

how many chromosomes do humans have

46, 23 pairs

what does DNA polymerase do?

DNA polymerase adds the deoxyribonucleotides to the "growing end" of the growing strand of DNA

How do the nucleotides bond?

the nucleotide has a triphosphate, one of the phosphates will bond with the OH group, leaving two phosphates free

how is the polymerisation reaction driven?

when the tri-phosphate breaks, energy is released

what is the first step of DNA replication?

opening the helix structure and separating the strands

what is the second step of DNA replication

what does helicase do?

helicase unwinds the double helix by breaking the hydrogen bonds that keep the strands attached

what does primase do?

Primase assembles a primer, a foundation at which replication can begin

what does polymerase do?

DNA polymerase wraps itself around that strand, beginning at the primer, and it attaches new nucleotides

what is the leading strand

the leading strand is 5'-3' continuously, natural direction

what is the lagging strand

not 5'-3' continuously, which is what polymerase needs to work, replication is in segments

Okazaki fragments

the 5' to 3' fragments of replicated dna on the lagging strand

1st step of PCR DNA amplification

Denaturation, to separate the dna strands 98 degrees

2nd step of PCR DNA amplification

Annealing, the primer attaches to the single strands

3rd step of PCR DNA amplification

Elongation, the polymerase works its magic, binding to its template, nucleotides are added and a copy is made

what kinds of primers are needed

both forwards and reverse primers are needed to make the dna copy highly specific

PCR cycle equation

Nm = N0 * 2^n

how does Small Molecule drug therapy work

small molecule drug therapy targets the protein that causes the mutant gene, without altering its shape

what stage of the central dogma does Small Molecule drug therapy affect

affects mRNA translation

how does RNA interference therapy work

small interfering RNA targets the segment of the sequence that is responsible for the production of the mutant gene. it binds to the sequence and slices it, stopping the translation of the mutant gene

what stage of the central dogma does RNA interference drug therapy affect

it targets translation, stopping it from occurring

how does gene replacement therapy work

viral vectors infiltrate the cell with a new DNA sequence that has been designed to cause reverse transcription of the viral RNA, which is then integrated into the original genome

what stage of the central dogma does gene replacement therapy affect

it affects the first stage of the central dogma, before transcription can take place

how does CRISPR- Cas9 gene editing therapy work

CRISPR-Cas9 introduces an enzyme called Cas9 that cuts down the virus gene with the aid of guide rna

what stage of the central dogma does CRISPR- Cas9 gene editing therapy affect

Cas9 enzyme cuts away the virus before transcription can take place, impacting the first stage of the central dogma

Protein inhibition is...

short term, safer, harder to produce

DNA inhibition is...

long term, non reversible, easier to produce since we know the building blocks

what is a challenge to RNA delivery?

RNA has a low bioavailability, most of it goes to kidney and liver, little goes to blood which is necessary

why not inject RNA therapy into vein?

the concentration of the therapy is too high, quick release isnt good

why is slow release good for therapy introduction

slow release maintains release profile, remains in the system longer

what does a viral vector do?

introduces specific DNA sequences to host cells

what is the shape of a viral vector

plasmid, circular

Original of replication (vv)

the DNA sequence that allows the start of replication in the plasmid by transcriptional machinery proteins

Antibiotic Resistance Gene (vv)

allows for the selection of plasmid containing bacteria, non plasmid containing bacteria will die.

Multiple cloning sites (vv)

short segment of DNA that has many restriction sites, easy insertion of dna

Promotor region (vv)

Transcription of key gene, RNA polymerase binds to the promotor

selectable marker (vv)

the antibiotic resistance gene for example, allows for selection in bacteria

insert (vv)

target gene that is cloned for the study

primer bonding site (vv)

single stranded DNA sequence used as an initiation point for PCR amplification or sequencing

Restriction enzyme

cut up the DNA sequence, used by bacteria to fight viral infection

straight cut (restriction enzyme)

blunt end

not straight cut (restriction enzyme)

sticky end

how are restriction enzymes used?

used to cut and paste various dna fragments together. based on the section of dna you want to remove, choose a specific enzyme to cut it out of the dna. mix with new dna you want to add

how is ligase used in dna cut and pasting

used to connect the segment of dna you want to paste into the dna as a whole. ligase is the enzyme, ligate is the process