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)
Cell-based cloning uses ____ to transfer DNA to host cells
process is based on ___
carry
allow it to
“vectors”
- Based on naturally occurring extrachromosomal replicons
- Carry target DNA as passenger into cells
- Most cloning vectors allow it to be replicated independently of host chromosome
Common cloning vectors
Plasmid - most common
Fosmid - sex chromosome
BAC - Bacterial artificial chromosome
YAC - Yeast artificial chromosome
eDNA
environmental DNA
animals constantly shedding DNA into aquatic or marine environment - can analyze to find evidence that organism is present in environment
yeast artificial chromosomes
DNA molecule that has a yeast origin of replication, a pair of telomeres, and a centromere
these features ensure that YACs are stable - replicate and seggregate in same way as yeast chromosome
useful - can cary fragments as large as 600kb
Bacterial artificial chromosomes
vectors constructed form F plasmid
can hold large fragments of DNA
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
expression vector
vector that contains sequences required for transcription and translation in bacterial cells
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
Cell-free (in vitro) gene cloning is
PCR
Polymerase Chain Reaction steps
1) 94-96C >30s → denature
2) 50-65C >3s → L and R primers anele to complementary sequences
3) 72 >30s → allows taq. pol. to bind to primer site and synthesize/extend
PCR copies double
every cycle
1 billion at cycle 30
1/2 billion at cycle 29
requirements for PCR
2 oligonucleotide (short DNA or RNA molecules, oligomers) primers complementary to opposite DNA strands. → primer for pol. to be active
Polymerase (Taq)
dNTPs (A,T, C, and G) → bases
Magnesium → req. by enzyme
Template DNA
Pros and Cons of PCR
Pro: Rapid isolation of rare target from complex mixture
e.g., 1.6 kb B-globin gene, 1.6 kb : 3200 Mb
Con: Disadvantaged by short lengths (<5 kb) and comparatively low yields
Amplicon
is the PCR product
- primers are integral to it (i.e., they contribute to its length (in nts)) → primers form then poly adds from there
- length (nts) does NOT depend upon the number of PCR cycles
- is double-stranded
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
PCR Applications
PCR mutagenesis
Real-time quantitative PCR
- Quantify DNA, mRNA
Whole genome amplification
- Add common double-stranded linker to all DNA fragments
Diagnostics
- e.g., Pathogen identification → amplify something specific to virus if amplification occurs than patent is positive
You have a 3 kb piece of DNA corresponding to a portion of the human dystrophin gene (involved in muscle function). You also have a small plasmid vector (for cloning in E. coli). Which of the following is likely to be common to both of these DNA fragments?
an origin of replication
introns
a selectable marker
restriction sites
None of the above.
an origin of replication → vector
introns
a selectable marker → vector
restriction sites → must have compatible restriction sitesto produce sticky ends
so ans. is restriction sites
A researcher has a plasmid vector that has been cut with a restriction enzyme that cuts after the first G in the sequence 5’GGATCC3’. The researcher has donor DNA that has been cleaved with a different enzyme that cuts after the first A in the sequence 5’AGATCT3’. (The paired complementary strand of each recognition site is not shown.) Based on this information you conclude that:
1- the cut DNA molecules will have single-stranded ends.
2- the vector and donor DNA will have complementary sticky ends.
3- the researcher will be able to form recombinant DNA molecules from these vector and donor DNAs.
4- portions of the cut plasmid and the cut donor DNA will be able to hybridize.
5- All of the answer options are correct.
5- All of the answer options are correct.
You want to amplify a specific DNA sequence from an influenza virus. Which of the following is necessary for the amplification of this DNA by PCR, but is not necessary for amplification by cloning?
1- presence of recognition sites for a specific restriction enzyme
2- mRNA from the virus so that noncoding DNA is not amplified
3- knowledge of the sequence of at least some parts of the virus DNA
4- presence of sticky ends for primer binding
5- None of the answer options is correct.
3- knowledge of the sequence of at least some parts of the virus DNA
You want to clone genomic DNA from a higher eukaryote, and your insert is 190 kb in size. The best choice for the cloning vector would be a:
BAC
fosmid
plasmid
any of the above
none of the above
BAC
Plasmid insert size
& copy number per cell
5-10kb
often high
Fosmid insert size
& copy number per cell
30-44kb
1-2
BAC insert size
& copy number per cell
up to 300kb
1-2
YAC insert size
& copy number per cell
0.2 to 2.0 Mb
(200kb to 2000kb)
low
