Lecture 13 Flashcards
Multiple olfactory receptor genes—and ____ variations on said genes—are involved in determining whether you have a talent for sniffing out asparagus pee
871 sequence
Duchenne muscular dystrophy
muscle weakness
duplications or deletions that cause frameshift mutations leading to premature stop condons that produce a truncated, nonfunctional dystrophin
Clinical case
What is differential diagnosis?
Differential diagnosis - the process of differentiating between two or more conditions which share similar signs or symptoms. - Parasite, ebola, malaria, flu etc.
How confirmed - blood work, tests
What is cloning
to make an identical copy
ex. dolly (first mammal that was cloned from an adult somatic cell)
Two types of cloning
Cell based - in vivo
Cell-free - in vitro
either way gene is amplified (or cloned) into many copies
In vivo cloning steps
1) Isolate and cut DNA (with restriction enzyme) from study organism
2) Join with a cloning vector
(self-replicating DNA from another source, e.g., virus or plasmid)
(Joining DNA from different sources creates recombinant DNA sometimes referred to as DNA “construct”)
3) Transfer recombinant DNA (construct) to host cells
4) Cell-based amplification occurs as cells replicate (and plasmids replicate within host cells)
Cloning (in vivo) evolved from
site specific nucleases (restriction enzymes) and DNA ligases
cloning into plasmid → (host organism) replicate cloned sequence
First step in cell based cloning is to
cut DNA into smaller pieces → gene of interest
(start w/ → molecular scissors b/c most DNA molecules are too big to manipulate)
Types of scissors (that fragment DNA into smaller pieces)
1) Physical
random shearing via, e.g., sonication (blast with high frequency sound waves)
2) Enzymatic → restriction enzymes
- naturally encoded in bacterial genome
- cut DNA at small, specific sequences
- bacterial defense against viruses
- bacterial DNA protected by methylation (restriction enzymes cut invading virus DNA but do not cut methylated bacterial DNA)
Is this a Genetic Palindrome?
5’-ATCCTA-3’
No
b/c backwards complement (5’ GATCCTAG 3’) not read same
Restriction enzymes (_____)
recognition sequence is _____
(endonucleases)
a short palindrome (reads the same on both DNA strands)
(→ arrow shows where enzyme cuts in sequence)
different enzymes cleave in ___
can be __ or ___
in different ways and in both strands
“staggered” or flush
staggered - asymmetrical cutting (sticky)
flush - in middle (blunt)
Which of the following sequences are (genetic) palindromes?
5’-G G A A G G-3’
5’-C C A T C C-3’
5’-G G G T T T-3’
5’-G A A T T C-3’
5’-A A A A A A-3’
5’-G A A T T C-3’
b/c
5’-G A A T T C-3’
3’-C T T A A G-5’
and when both 5’ - 3’
5’-G A A T T C-3’
5’-G A A T T C-3’
same
Staggered cleavage produces ___ ends
which can form ___
“sticky” (also called cohesive)
can form hydrogen bonds with complementary sequences from other sources
DNA from different sources can be joined if ____
the sticky ends are complementary
initial joining is via H bonding
Flush cleavage produces ____
“blunt” ends
can also be recombined → just less efficient
T4 DNA Ligase
covalently seals→ sticky (cohesive) or blunt (flush) ends
forms the phosphodiester bond (re-creates phosphodiester bond of the backbone)
more efficient → ligation of sticky ends
Separate enzymes with different recognition sequences may produce ___
which can then ___
causing loss of __
compatible sticky ends
Overhangs (sticky ends) can hydrogen bond
Loss of original recognition sequences →recombinant DNA cannot be re-cleaved by either enzyme (destroyed restriction site)
Mix-and-match useful for gene cloning
DNA fragments with blunt ends generated by different enzymes also can ____
be ligated together
OJ site destroyed (in this example)
Only ligations that reconstitute original recognition sequence can be re-cut by the original restriction enzyme
Only ligations that ___ can be re-cut by the original restriction enzyme
reconstitute original recognition sequence
Which enzymes leave compatible ends that can be ligated together:
HamHI 5’-G^GATCC
BglII 5’-A^GATCT
XbaI 5’-T^CTAGA
(^= where it cuts)
only HamHI and BgIII
(5’ → 3’ of one and 3’ → 5’ of other are compliments)
Common cloning vectors
Plasmid - most common
Fosmid - sex chromosome
BAC - Bacterial artificial chromosome
YAC - Yeast artificial chromosome
Ti plasmid
plasmid in soil bacterium - used to transfer genes to plants.
part of Ti plasmid DNA integrates into plant chromosomes - transcribed and translated to produce enzymes that support the bacteria
Cosmids
plasmids packaged in empty viral protein coats and transfered to bacteria by viral infection
essential features of cloning vectors
1) polylinker (at least one unique cloning site) → stretch of DNA that contains multiple restriction sites
2) origin of replication (ori) that is compatible with host
3) antibiotic resistance or other selectable marker (so can isolate cells w/ inserted DNA)
a polylinker is
a short segment of DNA which contains many restriction sites
aka a multiple cloning site
(color identification)
Insertion of foreign DNA in polylinker blocks ____ by ______
B-gal production by LacZ gene
polylinker is in the middle of LacZ
Intact polylinker doesn’t interfere with B-gal production by LacZ gene (polylinker is small and in-frame)
Gene product (B-gal) cleaves substrate (X-gal), =blue color
DNA insertion into polylinker blocks B-gal, =no color (white)
Plate of insertion of foreign DNA in polylinker
white____
blue ____
white - gene of interest (blocks b-gal)
blue- gene product (b-gal of LacZ gene) cleaves substrate (x-gal) causing blue color
Joining DNA with a cloning vector part ___ of ___ of cell based ____
part 1 of 2 of cell-based gene cloning
cloning vector with restriction site
cut with enzymes and connect w/sticky ends
now vector has gene of interest
Cloning via host cell replication
part 2 of 2 of cell-based gene cloning
Transformation of plasmid into cell
-not 100% efficient
-i.e., some cells don’t receive a plasmid
Amplification via replication
- Plasmids replicate
- Bacteria replicate
Antibiotic selection - to screen out non-transformed
Antibiotic selection
need because ___
need b/c process of transformation is not 100% efficient
antibiotic will kill bacteria that did not receive the vector
1) Design primers (5 nt long) that
will amplify a double-stranded
molecule containing the “red” part of the
sequence above.
2) How long (in nts) will the amplicon be?
1) write double strand
Always recall that DNA is synthesized 5’>3’, on both strands (Primers therefore are always written 5’>3’)
SOLUTION: Primer pairs would be written as:
5’-GTGAG and 5’-ATAGA; Amplicon = 21nts (2x5(nuc primers)+11(middle)= 21nts
Primers are always written as
5’>3
