BIMM 100: Week 3

purpose of polymerase chain reaction (PCR)

synthesize millions of copies of a specific DNA fragment (template dsDNA, primers, DNA polymerase, dNTPs)

PCR reaction steps

denaturation (94C, 30s), primer annealing (30-65C, 30s), elongation/DNA synthesis (65-75C, 2-5min), repeat 25-40 times

taq DNA polymerase

stable at high temperatures

DNA electrophoresis

allows the separation of DNA by size

Steps for DNA Electrophoresis

DNA samples loaded into wells of gel agarose tray, electrodes turned on and fragments travel, DNA fluorescent dye added

DNA is negative because of its…

phosphate group

Purpose for Southern Blot

can identify a specific DNA sequence in a complex mix

Steps for Southern Blot

endonucleases cut DNA, pieces separated by size by gel electrophoresis, alkaline solution separates dsDNA into ssDNA, transfer to membrane, complementary DNA with probe binds to desired sequence

Steps for Sanger Sequencing

template DNA, primer DNA, DNA polymerase, dNTPs, ddNTPS (randomly terminate synthesis)

Purpose for Sanger Sequencing

sequencing by synthesis, every possible fragment length is synthesized to reveal sequence

Limitation of Sanger Sequencing

cannot read past 1000 nucleotides, solution is to identify overlaps between different regions

Steps for Next-generation reversible terminator sequencing

Add fluorescently labeled 3’-OH blocked dNTPs, wash out unincorporated nucleotides, read fluorescence, remove label and blocking group

Unlike sanger sequencing, next-generation sequencing can…

synthesize a single molecule

Key development #2 of NGRTS

millions of genome fragments can be simultaneously sequenced in a tiny space

nucleosome

structural subunit of chromatin (~150 bp), condenses DNA length

chromatin

the entire complex of histones and DNA

DNAse

nuclease that degrades histones and separates DNA

heterochromatin

condensed chromatin, generally inactive

euchromatin

decondensed chromatin, generally active

How do you determine if a specific chromatin region is in a condensed or decondensed area?

DNAse treatment + PCR/Southern Blot, measures how easy it is for DNAse to destroy fragment

gene

the entire DNA sequence needed to make a functional gene product

coding genes

produce mRNAs that encode proteins

non-coding genes

produce other RNAs (ribosomal, transfer, telomerase RNA component)

promoter

regulates the rate of transcription of the RNA coding region

solitary genes

found only once in the genome

duplicated genes

multiple copies that are close, heavily used and usually found in same region

heavily used duplicate gene products

rRNAs, tRNAs, snRNAs (splicing)

Most protein-coding genes are…

solitary

monocistronic vs polycistronic

1 mRNA = 1 protein vs 1 mRNA = several proteins

Eukaryotic pre-mRNAs are…

capped (5’ head), spliced (remove introns, polyadenylated (3’ poly-A tail)

simple genes

coding genes that produce one mRNA, one product

complex genes

produce multiple mRNAs via alternative splicing (so multiple products)