exam 2: DNA at high resolution; studying genomes Flashcards
How do genetics “dissect” genomes?
- restriction digests: enzymes that cut DNA
- electrophoresis: use an electrical field to separate charged particles by size
- molecular cloning: cutting the DNA of one organism; out into diff pieces of gene in another organism
–library construction/ screening - polymerase chain reaction: 1 genome and make many copies
- sequencing: determine the order of DNA while DNAs being synthesized
restriction digest
-cut dna into smaller pieces by digesting w REs
-cut both DNA strand thru sugar phosphate backbone
-only cut at specific DNA sequeces (highly specific-recognize 1 sepecfic sequence; not random)
each recognition sequence have a diff RE
why dont bacteria cut up their own DNA?
-methylate RE sites
—> highly specific: some bacteria dont have the sequence
—-> add methyl groups on the sequece, which blocks the enzyme
restriction enzymes: palindromes
most Re recognize 4-6 base pair palindromes
palindromes: sequence that is identical on both strands when read in 5’ to 3’ direction
-contain mirror image of complement
5’GAATTC3’
3’CTTAAG5’
complement
5’GAA|TTC3’
3’CTT|AAG5’
*RE-cutting properties: Blunt cutters
aka polish-end
-cut at the same point on BOTH strands all the way thru the backbone
*RE-cutting properties: Overhang cutters
aka sticky ends
-cut at diff places thorugh backbone leaving a single short strand overhang
why cutting 2 separate places?
DNA is antiparrallel
5’G. GATCC3’
3’CCTAG G5’
How are RE’s cut sites estimated
- assume all bases are in equal amounts (25% each)
- assume bases are distributed randomly
-neither assumption usually true, but this process will give rough estimate
What’s the frequency of RE cutting when it recognizes 4 base pair sequence? 6 base pair sequence
product rule
4 base pairs: (1/4)(1/4)(1/4)(1/4)=1/256
6 base pairs: (1/4)(1/4)(1/4)(1/4)(1/4)(1/4)=1/4096
smaller sequence=high probability of being cut (less specific)
Electrophoresis
-separation of charged particles in electric field
-can use w DNA, RNA, or protein (+,-,N)
DNA in agarose gel:
matrix of agarose that forms millions of microscopic pores
* DNA added to agarose matrix
* When electric current applied, negatively charged DNA migrates to the positive pole bymoving through pores
* Big pieces of DNA move slower, little
fragments move faster
Agarose: no less than 30-50
polyacrylamide: <30
eletrophoresis: agarose and polyacrylamide gel
Agarose: no less than 30-50
polyacrylamide: <30
agarose: polysaccharide from sea weed
-dry, power form: loose, unconnected chain
-heated, then cooled: twist + form helices
polyacrylamide: polymer of acrylamide molecules held together
by bis-acrylamide bridges.
cloning
use this method after cut up the genome inorder to isolate a specific fragment
how? clone DNA fragments via molecular cloning
can you isolate a specific region of DNA wo cutting RE?
-via PCR
Molecular cloning
Clone individual DNA fragments from a
complex mixture into living cells, one
fragment at a time
1) ligation
2) transformation
molecular cloning: 1. ligation
Ligation: physically bind DNA fragment into a
vector using the enzyme ligase
-vector(plasmid): a specialized DNA sequence that
1)can enter a living cell (host)
2)detectable by an investigator (usually its antibiotic resistance)
3) able to replicate itself while living in the host
most vectors are bacterial plasmids (small, circular, extrachrosomal pieces of DNA)
*host cells are often bacteria (more likely to make copies, instead of destroying it)
what does ligation require? (molecular cloning)
requires “compatible” fragment and vector ends (biochemically attracted to each other)
- How is this accomplished? Cut fragment and
vector with same RE (requires vector to only
have 1 site recognized by specific RE) - Only 1 fragment will be ligated into 1 vector
(recombinant DNA)
=>inefficient process (1 fragment & 1 vector @ a time)
molecular cloning: tranformation
-separate vectors that have diff fragment
Mix recombinant DNA (vector + fragment)
with specialized host cells (usually bacterial)
that are “competent”
1.Able to take up foreign DNA
2.One host cell will only take up one vector
- When host cell divides, recombinant DNA is
copied and passed to new cell (cloned
molecular cloning: transformation: lab
Grow transformed cells in liquid media then plate on
plates that contain antibiotic
* Only cells that have taken up the vector (that gives
antibiotic resistance) will survive
* An individual surviving cell will divide on the plate
many times to produce thousands of exact copies
(including exact copies of vector and ligated
fragment)
* So many cells you can see “colony” of bacteria
* Can grow colony to produce even more “clones”,
then isolate vector and its ligated fragment
*bacterial colonies: antibiotic, only cells with vector survive
-no 1 colony is gentically identical
-diff bc they have diff recombonatants
the purpose of molecular cloning? what can u do w a cloned fragment?
- express polypeptide (if cloned fragment is protein-coding)
- intentionally mutate sequence (to figure out the function- get an idea of what that gene can do)
- sequence the DNA fragment
- make a library
*genomic library
entire genome of organism
cut up into fragments and cloned into vectors
an ideal genomic library:
1 copy of every chromosome cut into fragments
that are individually cloned into vectors (DNA from 1 gamete)
*cant: requires too much DNA…why?
1. inefficiency process (1 fragment for 1 DNA)
2. polarity of water
=lose things along the way; need to start out w way more
-to account for the fact that sample will be lost along the way
how to make genomic library?
cut DNA w REs, cloning fragments into vectors, and transform the recombinant DNA
how is genomic library useful
-contain >1 copy of some sequences bc u begin with so many nuclei
-contain alll forms of DNA
(coding, non-coding, telemores…)
useful:
-can fragment and put into GenBank
-comparison
-study DNA from extinct animals to bring them back
-study telemere of the entire genome of animals
*complementary library (cDNA library)
-has to do with protein coding DNA (actively being expressed as protein)
-start w mRNA to make DNA
-looking at protein coding sequences that are actively expressed during the time the making of the lib
the process of making cDNA lib
-contain DNA fragments derived from mRNA
why mRNA? already transcribed, will get translated
_use mRNA as a template, use reverse transcriptase to make the template (RNA-DNA copy)
ways to make cDNA libs
- make cDNA lib from same organism but 2 diff tissue sources
how useful? to study what genes are expressed
mRNA brain vs mRNA liver
-to see what genes are specialized
-to see what genes are housekeeping genes: essential genes - make cDNA lib from same organism but exposed to 2 diff environments
how useful? looking for diff in gene expression
what genes is being expressed due to environmental differences?
-normally not expresed until u change the encironment
when you u use genomic lib instead of cDN lib
if u want to study the entire genome or non-coding region of DNA (promoter, telemere, intron,…
