DNA Analysis and Sequencing and Epigenetics

PCR

Polymerase Chain Reaction - used to amplify a specific sequence of DNA

Gel Electrophoresis

Used to visualize the results of PCR

Sanger Sequencing

It needs the same things as regular DNA replication, but uses ddNTPs as well as dNTPs

4 separate reactions are run in order to figure out the location of the bases

dNTPs

Has a hydroxyl group that the next base can bind to

If you only added dNTPs, the gel would show up as one band of the full DNA sequence

ddNTPs

Doesn’t have the hydroxyl group and no base will bind to it

If you only added ddNTPs, you won’t know the position of the very first base

dNTP:ddNTP Ratio

100:1

Automated Sanger Sequencing

A computer reads the sequence, and it comes up as a colored chart where each color corresponds to a base

SNPs

Single Nucleotide Polymorphism - the origin of these polymorphisms is determined by comparing the human genome with those of other species (If the sequences are different in two humans, then it is a SNP)

SSRs

Simple Sequence Repeats - 1-10 base repeats occurring 10 to over 100 times, usually found in non-coding regions, arise much more quickly than SNPs

Polymerase Slippage Model

During DNA replication, the strand will make a loop and once it straightens out, a bunch of DNA will be replicated, leading to these repeats

Microsatellite Genotyping

You can design PCR primers that are unique to one locus and can base pair on either side of the repeated portion, these primers can be used for every individual in the species

Epigenetics

Changes the function of a gene without changing the DNA sequence

Histones

Proteins that organize the genetic material, pack DNA into chromatin which make up chromosomes

Euchromatin

Condenses and decondenses throughout the cycle, contains DNA that is actively transcribed, devoid of repetitive sequences

Heterochromatin

Remains condensed throughout the cycle, typically transcriptionally inactive, can have repetitive sequences and always be condensed or condensed in some stages

Chromatin State

DNase hypersensitive sites can be formed when histones are moved by chromatin remodeling complexes

Histone Tail Modifications

Acetylation or Methylation

Acetylation

Histone acetyltransferases are added to the histone tails

Prevents close packing of nucleosomes (opens everything up)

Favors euchromatin

Reversed by histone deacetylases

Methylation

Histone methyltransferases are added to histone tails

Closes everything up

Favors heterochromatin

Reversed by histone demethylases

Methylated Cytosines

Happens in the promoter regions of genes (not the gene itself) and turns off the expression of genes, added by DNMTs

CpG Islands

Islands with high concentrations of CpG dinucleotides

Usually unmethylated because an activator is binded to the island and is blocking the DNA methyltransferase

RNA Interference

When double-stranded RNA silences gene expression, leading to degradation or inhibition of the target mRNA