Lecture 29: Genomics Flashcards
4 methods used in nucleic acid biochem and genome analysis
- gene cloning
- DNA sequencing
- polymerase chain reaction (PCR)
- microarrays
genome
complete set of DNA sequences contained w/in single organism
eukaryotic DNA
mostly in nucleus, but can be in mitochondria or chloroplasts
2 copies of each gene in every cell
bacterial DNA
contain plasmids (extra chromosomal pieces of DNA) chromosomes are circular
Centra dogma
DNA–>RNA–>Protein
chromosomes in eukaryotes vs. prokaryotes
eu: 1+ linear chromosomes, in nucleus
Pro: circular chromosomes, no nuclear envolope
complete genome replicated in each cell division=two cells (in both)
human chromosomes
23 pairs, 22 autosomal, XY determine gender
Is the human karyotype (chromosome spread) from a male or female? SLIDE 4
male
condesnation of chromosomes
histones: form nucleosome particles
these are then packaged too
transcription of segments of DNA into RNA by…
polymerases
RNA Pol 1
generates single strand RNA product with protein coding sequences (exons)
scattered among introns (non-coding)
mRNA created when…
enzymes remove non-coding sequences to make pre mRNA into mRNA
bioinformatics
used to predict locations of genes w/in regions of genomic DNA using computers
find sequences you think do certain things
BLAST is common
look for pieces of DNA across organisms
can show matches between DNAs of different organisms to give clues to functions
where can coding regions be?
ON EITHER STRAND of DNA
can be on “top or bottom” gene
Werner’s syndrome
premature aging, die from things caused by old age, but young
mutation in DNA helicase; it unwinds the defective DNA
damage to DNA not properly repaired.
number of genes vs. number of base pairs
larger genome doesn’t mean you have more genes
How we use genomic info to understand human disease states
known sequences of normal and abnormal genomes
we can see what may give rise to a particular disease
What is the biochemical defect in the WRN mutant protein
truncated protein, stops being made early because of a wrongly placed stop codon
what are in genomes?
coding and non coding sequences
How we ID’d Werners system
focused on a protein that turned out to be DNA helicase (only needed during repair)
found that there was a mutation in Werner’s patients that an argenine was instead a stop codon
left with non-functional protein
where is it more frequent to see differences in individual’s genomes?
in non-coding regions
not gene mutations in proteins
These are DNA polymorphisms, and are useful genetic markers
most common: SNP (single nucleotide polymorphisms) and STR (short tandem repeat) polymorphisms.
Give 3 examples of how DNA polymorphisms are used for genetic identity
paternity testing
crime scene analysis
following the path of a virus to find start of outbreak
DON”T have to sequence whole genome
Most common polymorphisms
most common: SNP (single nucleotide polymorphisms) and STR (short tandem repeat) polymorphisms.
basically how STRs and SNPs work
compare + and - states, they differ by just one nucleotide
recognized by the enzyme: restricted endonuclease
It cuts a specific sequence at a specific site. changing by one nucleotide means this doesn’t happen
How we can read out STRs and SNPs work
cuts are different, we have them read out
one sequence will be cut, we separate on gel based on size
bands look different
GO BACK AND LISTEN TO 12
GO BACK AND LISTEN TO 12
bacterial plasmid
extra chromosomal piece of circular DNA in bacteria
Gene Cloning
using bacteria to isolate and amplify specific gene sequences
3 types of biochem molecs needed for recominant DNA technology
- use bacterial plasmids, get copied by bacteria
- restricted endonucleases: protection for bacteria against invading DNA
- DNA Ligase
Restriction enzymes
used to protect bacteria from viral incection
Methylated bacterial DNA not cleaved by restriction enzymes
Why cleave? why methylate?
invading DNA cleaved and destroyed before methlateion
id as self or non self
nuclease can’t cleave methylated DNA, can only cleave unmethylated
how restriction enzymes work
bind specific DNA sequences 4-10 nucleotides long (double standed)
catalyze DNA cleave reactions
most are homdimers, so recognition sequence is palindrome (read it forward or backward, its the same)
what cleavage can generate
staggered ends
blunt ends
ends determine how easy it is to handle and insert new DNA (staggered is easier for insertion)
how to do gene cloning
take the gene of interest out of its original context
put it in bacterial vector–> protein expression for study
open the vector/plasmid
they have the same restriction sites and EcoR, so the cuts match up
linearize the cuts
paste in DNA
close vector
Ligase seals the break in the backbone
DNA Ligase
uses ATP to seal break in backbone
Multiple cloning sites (MCSs)… many different types of restriction enzyme sequences… WHY???
to facilitate cloning of lots of different types of genes of interest
what do we do after we put the gene in the bacteria???
DNA is isolated by lots of steps. purify it. pretty quick. (check size)
maybe isolate again from gel
transformation
plasmid propagated
transformation
what we call putting the DNA in the bacteria system
Result of gene cloning?
make a lot of particular kind of protein for study of things like:
turn over rate, substrate specificity, etc
DNA Sequencing
use fluorescent probes to get a read out of nucleotides
add some dideoxynucleotides, they are terminators for chains because they lack an OH group
whenever a dideoxidenucleotide is incorporated, chain stops growing (very small amount of these)
all pieces differ by one fluoresecent dideoxynucleotide. separate by size
COLOR TELLS THE COMPLIMENT of the DNA
Polymerase Chain Reaction (PCR)
amplify DNA segments using primers on each of 2 DNA strands
heat then anneal single strand primers
in virto synth reaction, generation of 2 new DNAs
each time you anneal, replicate, denature, you get more product (exponentially)
heat kills the enzyme for PCR, so we use…
TAC polymerase because it lives in hot environments
Why does it actually take over 25 cycles to reach 1 mllion molecules, since the math says it should be 20?
the enzyme is not 100% efficient.
High Throughput Microarrays… why?
DNA sequences dont say anything about genome function or regulation
real question: Which genes are expressed in what tissues when?
do expression patterns differ between health and diseased indivs? can we use these patterns for diagnosis?
High Throughput Microarrays
covalently link single strand DNA sequences to a literal chip
incubate single strand RNA w/ DNA on slide under hybrid conditions
label RNA fluorescently
determine RNA-DNA hybrid position on array
pattern given by microarray…
tells whethere gene is off or on AND
how much on or off they are
laser disection
shows pathologists whats going on in the tumor as compared to elsewhere
RNA Seek
next generation of microarray
sequence everything in the cell
computer counts.
