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Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from
1,000 to 8,000 kilobase pairs.
Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule.
Bacterial Genome: The collection of all of the genes present on the bacterias
chromosome or its extrachromosomal genetic elements.
Only in RNA,
not DNA
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
Restriction Enzymes
Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restriction
enzymes.
Cut up foreign DNA that invades the cell.
Type II and III restriction enzymes cleave DNA chains
at selected sites.
Enzymes may recognize 4, 6 or more bases in
selecting sites for cleavage.
An enzyme that recognizes a 6-base sequence is
called a "six-base cutter.
No ATP requirement.
Recognition sites in double stranded DNA have a 2-fold
axis of symmetry a palindrome.
Cleavage can leave staggered or "sticky" ends or can
produce "blunt ends.
Don't nod
Dogma: I am God
Never odd or even
Too bad I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Dennis sinned
EcoRI
BamHI
DpnI
HindIII
BglII
PstI
Sau3AI
KpnI
5AAGCTT35AAGCTT3
3TTCGAA5 3TTCGAA5
5GGTACC3
3CCATGG5
3CCATGG5
5CCCGGG3
3GGGCCC5
5AAGCTT3
5AAGCTT3
3TTCGAA5
3TTCGAA5
CCCGGG
AAATTT
CCCTTT
AAAGGG
Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT) can be recut by SmaI or DraI.
Mixed ligation products (CCCTTT + AAAGGG) cannot be re-cut by SmaI or DraI.
GATCT
A
No longer
palindromic, so not
cut by BamHI or BglII
Ampr
Ori
pBR322
4361bp
Tetr
LacZ
MCS
pUC18
Ampr
Ori
Cloning Vectors
Older cloning vector
Ampr
Ori
pBR322
4361bp
Tetr
pUC18
Ampr
Ori
Chimeric Plasmids
Named for mythological beast
(chimera) with body parts from several
creatures.
After cleavage of a plasmid with a
restriction enzyme, a foreign DNA
fragment can be inserted.
Ends of the plasmid/fragment are
closed to form a "recombinant
plasmid.
Plasmid can replicate when placed in a
suitable bacterial host.
CF
TR
LacZ
MCS
pUC18-hCFTR
Ori
Ampr
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
normal
dNTP
ddNTP
H
A nucleotidespecific stop in
DNA synthesis
NCBI.nlm.nih.gov/genome/guide/human/index
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
HpaI Digest
Variants
Nor3
mal 1 2
70% of carriers of the sickle cell
gene have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI
fragment
Mutant
Pro Val
CCT GAG
DdeI site
CCT GTG
no DdeI site
AS AS SS AA
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
94C
37-65C
70-75C
3
5
5
2 original strands.
PCR in Medicine
PCR in Forensics
Crucial forensic evidence may be present in very small quantities.
often too little material for direct DNA analysis.
but PCR can generate sufficient DNA from a single cell.
PCR also possible on extensively degraded DNA.
examples include DNA from single dried blood spot, saliva (on cigarette
butt), semen, tissue from under fingernails, hair roots.
Other advantages of PCR in forensic science are:
relatively simple to perform and simple to standardize.
results obtainable within 24 hours.
The major legal problems with PCR are the potential for crosscontamination between samples and the complexity of explaining what
the results mean to the jury.
DNA typing is only one of many pieces of evidence that can lead to a
criminal conviction, but it has proved invaluable in demonstrating innocence.
Dozens of cases have involved people who have spent years in jail for
crimes they did not commit until PCR exonerated them.
Even when evidence such as semen and blood stains are years old, PCR
can make unlimited copies of the tiny amounts of DNA remaining in the
stains for typing.
Conclusions
Background
DNA cloning
DNA sequencing
Detection of disease genes
Polymerase chain reaction (PCR)
PCR basics
PCR in medicine
PCR in forensics
Questions?