exam mol bio

is the synthesis of a new DNA

molecule from an existing DNA molecule

DNA replication

The 3 DNA Replication Hypothesis

conservative semi conservative dispersive

One molecule would be entirely parental DNA, and the other would be entirely new DNA.

conservative

the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand

semi conservative

unwinds dna

helicase

semi conservative Process:

helicase unwinds dna then each strand serve as template for dna replictation. Dna poltmerase then sythesizes new strands in 5’ to 3’ direction after two dna strands are form

A special type of semi-conservative replication seen in circular bacterial DNA.

theta replication

DNA opens at the

origin of replication

theta replication seen in

e coli and plasmids

theta replication process

dna opens at origin of replication and then forms a replication bubble then repilcation starts bidirectionally until two circles appear

DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA

dispersive model

DNA Replication Follows the

semiconservative model

used equilibrium density gradient centrifugation experiments to demonstrate that DNA replication is semiconservative

Matthew Meselson and Franklin Stahl

DNA replication can be divided into three phases:

initiation elongation termination

DNA replication starts at specialized sites called origins of

replication and moves away from an origin in both directions,

creating a structure known as a replication bubble

initiation

DNA replication starts at specialized sites called

origin of replication

when dna replication moves away from origin what does it create

replication bubble

initiator proteins bind to the replication origin, a base-pair sequence of nucleotides known

oriC

when replication of DNA begins, these sites are referred to as

replication fork

enzyme unwinds the double helix and exposes each of the

two strands so that they can be used as a template for replication

helicase

synthesises a small RNA primer, which acts as a ‘kick-starter’ for DNA polymerase.

dna primase

DNA primase responsible for

creation and expansion of new strands of DNA.

prevents the DNA double helix ahead of the replication fork from tight wounding

topoisomerase

extend the primer by adding free nucleotides to the 3’ end new strand

dna polymerase 3

DNA synthesis occurs in fragments, what are these fragments called

okazaki fragments

removes all RNA primers in the lagging strand

exonuclease

The nicks that remain after the primers are replaced get sealed by the

ligase

is the process by which the information in a gene is used to synthesize functional gene products, such as proteins or RNA molecules.

gene expression

Epigenetic Regulation is

DNA methylation and histone modification affect gene accessibility.

is the study of changes in gene expression that do not involve alterations to the DNA sequence itself.

epigenetics

these epigenetics changes are due to

environemtal factors, lifestyle choices, and experiences

Proteins that bind to specific DNA sequences to regulate transcription.

transcription factors

Activate gene transcription.

Enhancers:

Suppress transcription.

Repressors:

What controls transcriptional regulation?

Transcription factors enhance/repress RNA polymerase activity.

•The process of copying the sequence of one strand of DNA, the template strand

•mRNA copies the template strand

Transcription

the side of DNA that will be used to create an mRNA strand

template strand

Transcription requires

rna polymerase

rna polymerase reads the template in what direction

3’ to 5’

List the three eukaryotic RNA polymerases and their roles.

pol1synthesizes rRNA precursors.

pol2 synthesizes mRNA precursors.

pol3 synthesizes tRNA precursors

First phase of transcription is

initiation

Describe the four steps of transcription initiation.

Closed promoter complex formation.

Conversion to open promoter complex.

Polymerizing first 10 nucleotides.

Promoter clearance (stable RNA-DNA hybrid)

is one of the two DNA strands in a gene. It has the same sequence as the mRNA (except that DNA has thymine (T) instead of uracil (U) in RNA).

coding strand

are DNA sequences that provide signal for RNA polymerase, and they are where RNA polymerase binds.

Promoters

promoters other name

tata box

Promoters Bacterial elements:

• 10 box (Pribnow box)

• -35 box

  • TSS (transcription start site)

How do coding and template strands differ?

coding matches the mrna while template is complementary

After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed

elongation

Compare intrinsic and rho-dependent termination

Intrinsic forms hairpin loop while rho binds to mrna

segments of DNA that code for proteins, are then rejoined by the enzyme ligase

Exons

THE IMPORTANCE OF DNA EXTRACTION

To obtain high-yield and high-quality genomic DNA from any selected population of samples

The type of sample afffects

how much the yield and purity of dna

DNA Extraction Methods

1

Produces high-quality DNA but involves toxic chemicals.

Phenol-Chloroform Extraction:

Easy to use and yields high-purity DNA.

Silica Column-Based Kits:

Automated, scalable, and provides high-quality DNA.

Magnetic Bead-Based Extraction:

Effective for removing polysaccharides and secondary metabolites.

ctab methods

Factors Affecting DNA Yield and Quality

2

Proper cell lysis ensures

maximum dna release

Removes unwanted RNA contamination.

rnase treatment

Ensures pure DNA without inhibitors.

Protein and Lipid Removal:

Using the right ____ can optimize DNA recovery.

buffer

purity of pure dna

1.8 to 2.0

in gel electrophoresis high-molecular-weight bands indicate

good quality of sample

for long term stability dna should be stored at

-20 to -80c

Issues to consider in choosing particular samples:

a. Technical requirements

b. time efficiency

c. cost-effectiveness of the chosen method

Why dna purification Matters in Molecular Diagnostics?

in pcr leads to failed amplification in ngs may lead to sequencing failure in forensic could lead to inconclusive results

one of the most commonly used method for isolation and enrichment of human mononuclear like peripheral blood

Ficoll density gradient centrifugation

Ficoll density gradient centrifugation sample needed

whole blood

Ficoll density gradient centrifugation medium used

ficoll hypaque

ficoll density isolates

mitochondria, nuclei, or other organelles.

ficoll density primary purpose

isolating dna, proteins and viruses

Quick Extraction Through Proteinase K and Phenol blood is mixed with

Tris, EDTA, sodium dodecyl sulfate (SDS), MgCl2, and proteinase K

why is Dry Blood Spot Preparation performed

causes minimal risks to the participant like children and infants

DNA extracted from blood spots stored for up to

25 years

A hair with root is incubated at 95°C for 10min in NaOH buffer, and the supernatant is subjected to DNA purification after centrifugation

Genomic DNA Purification From Hair

The buccal swab samples are first suspended in lysis buffer that includes Tris, EDTA, SDS, and proteinase K.

Genomic DNA Purified From Saliva

Genomic DNA Purified From Saliva sample is incubated for

1-3 hours at 56c

The urine specimen is inverted or swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation.

Genomic DNA Extraction From Urine Sample

Genomic DNA Extraction From Urine Sample incubated for

2h 56c

method used to harvest cells

centrifugation

for cell lysis of gram pos cells

Use lysozyme or mechanical beating

for cell lysis of gram neg cells

Easier to lyse using detergents

for cell lysis of fungi cells

Require enzymatic digestion

for cell lysis of dna virusees

Use proteinase K digestion.

break the lipid barrier surrounding cells by solubilizing proteins and

disrupting lipid-lipid, protein-protein, and protein-lipid interactions.

detergents

It is frequently used to release DNA from cells by a boiling treatment, at the same time protectingthe DNA from the boiling effects with resin beads.

Chelex-100 Method

A method where DNA binds to silica particles in diatomaceous earth under chaotropic conditions

Diatomaceous Earth Extraction

a diatomaceous earth (diatomite) is a form of silica composed of the siliceous shells of unicellular aquatic plants of microscopic size.

Kieselguhr

positively charged diethylaminoethyl cellulose (DEAE) groups

on the resin’s surface and negatively charged phosphates of the DNA backbone

Anion-Exchange Extraction