Lecture 29: Genomics

Question 1

4 methods used in nucleic acid biochem and genome analysis

Answer 1

1. gene cloning
2. DNA sequencing
3. polymerase chain reaction (PCR)
4. microarrays

Question 2

genome

Answer 2

complete set of DNA sequences contained w/in single organism

Question 3

eukaryotic DNA

Answer 3

mostly in nucleus, but can be in mitochondria or chloroplasts
2 copies of each gene in every cell

Question 4

bacterial DNA

Answer 4

contain plasmids (extra chromosomal pieces of DNA)
chromosomes are circular

Question 5

Centra dogma

Answer 5

DNA–>RNA–>Protein

Question 6

chromosomes in eukaryotes vs. prokaryotes

Answer 6

eu: 1+ linear chromosomes, in nucleus
Pro: circular chromosomes, no nuclear envolope
complete genome replicated in each cell division=two cells (in both)

Question 7

human chromosomes

Answer 7

23 pairs, 22 autosomal, XY determine gender

Question 8

Is the human karyotype (chromosome spread) from a male or female? SLIDE 4

Answer 8

male

Question 9

condesnation of chromosomes

Answer 9

histones: form nucleosome particles

these are then packaged too

Question 10

transcription of segments of DNA into RNA by…

Answer 10

polymerases

Question 11

RNA Pol 1

Answer 11

generates single strand RNA product with protein coding sequences (exons)
scattered among introns (non-coding)

Question 12

mRNA created when…

Answer 12

enzymes remove non-coding sequences to make pre mRNA into mRNA

Question 13

bioinformatics

Answer 13

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

Question 14

where can coding regions be?

Answer 14

ON EITHER STRAND of DNA

can be on “top or bottom” gene

Question 15

Werner’s syndrome

Answer 15

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.

Question 16

number of genes vs. number of base pairs

Answer 16

larger genome doesn’t mean you have more genes

Question 17

How we use genomic info to understand human disease states

Answer 17

known sequences of normal and abnormal genomes

we can see what may give rise to a particular disease

Question 18

What is the biochemical defect in the WRN mutant protein

Answer 18

truncated protein, stops being made early because of a wrongly placed stop codon

Question 19

what are in genomes?

Answer 19

coding and non coding sequences

Question 20

How we ID’d Werners system

Answer 20

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

Question 21

where is it more frequent to see differences in individual’s genomes?

Answer 21

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.

Question 22

Give 3 examples of how DNA polymorphisms are used for genetic identity

Answer 22

paternity testing
crime scene analysis
following the path of a virus to find start of outbreak
DON”T have to sequence whole genome

Question 23

Most common polymorphisms

Answer 23

most common: SNP (single nucleotide polymorphisms) and STR (short tandem repeat) polymorphisms.

Question 24

basically how STRs and SNPs work

Answer 24

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

Question 25

How we can read out STRs and SNPs work

Answer 25

cuts are different, we have them read out one sequence will be cut, we separate on gel based on size bands look different

Question 26

GO BACK AND LISTEN TO 12

Answer 26

GO BACK AND LISTEN TO 12

Question 27

bacterial plasmid

Answer 27

extra chromosomal piece of circular DNA in bacteria

Question 28

Gene Cloning

Answer 28

using bacteria to isolate and amplify specific gene sequences

Question 29

3 types of biochem molecs needed for recominant DNA technology

Answer 29

1. use bacterial plasmids, get copied by bacteria 2. restricted endonucleases: protection for bacteria against invading DNA 3. DNA Ligase

Question 30

Restriction enzymes

Answer 30

used to protect bacteria from viral incection | Methylated bacterial DNA not cleaved by restriction enzymes

Question 31

Why cleave? why methylate?

Answer 31

invading DNA cleaved and destroyed before methlateion id as self or non self nuclease can't cleave methylated DNA, can only cleave unmethylated

Question 32

how restriction enzymes work

Answer 32

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)

Question 33

what cleavage can generate

Answer 33

staggered ends blunt ends ends determine how easy it is to handle and insert new DNA (staggered is easier for insertion)

Question 34

how to do gene cloning

Answer 34

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

Question 35

DNA Ligase

Answer 35

uses ATP to seal break in backbone

Question 36

Multiple cloning sites (MCSs)... many different types of restriction enzyme sequences... WHY???

Answer 36

to facilitate cloning of lots of different types of genes of interest

Question 37

what do we do after we put the gene in the bacteria???

Answer 37

DNA is isolated by lots of steps. purify it. pretty quick. (check size) maybe isolate again from gel transformation plasmid propagated

Question 38

transformation

Answer 38

what we call putting the DNA in the bacteria system

Question 39

Result of gene cloning?

Answer 39

make a lot of particular kind of protein for study of things like: turn over rate, substrate specificity, etc

Question 40

DNA Sequencing

Answer 40

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

Question 41

Polymerase Chain Reaction (PCR)

Answer 41

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)

Question 42

heat kills the enzyme for PCR, so we use...

Answer 42

TAC polymerase because it lives in hot environments

Question 43

Why does it actually take over 25 cycles to reach 1 mllion molecules, since the math says it should be 20?

Answer 43

the enzyme is not 100% efficient.

Question 44

High Throughput Microarrays... why?

Answer 44

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?

Question 45

High Throughput Microarrays

Answer 45

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

Question 46

pattern given by microarray...

Answer 46

tells whethere gene is off or on AND | how much on or off they are

Question 47

laser disection

Answer 47

shows pathologists whats going on in the tumor as compared to elsewhere

Question 48

RNA Seek

Answer 48

next generation of microarray sequence everything in the cell computer counts.