Escolar Documentos
Profissional Documentos
Cultura Documentos
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Introduction to Cloning and Recombinant
Technology: Lecture Outline
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
DNA is the genetic material of most
organisms (from bacteria to humans)
Plasmid
Chromosome: Most bacteria have one circular DNA chromosome ranging in size from
1,000 to 8,000 kilobase pairs.
Bacterial Genome: The collection of all of the genes present on the bacteria’s
chromosome or its extrachromosomal genetic elements.
Basics: Nucleotides are the
building blocks of DNA
Only in RNA,
not DNA
Deoxyribonucleic acid (DNA) is a long
double-stranded chain of nucleotides
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
What Does It Mean: “To Clone”?
• 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.
Recognition/Cleavage Sites of Type II
Restriction Enzymes
Examples of Palindromes:
• SmaI
-CCCGGG-
• SmaI
-CCC GGG-
-AAATTT-
• DraI -AAA TTT-
-CCCTTT-
-AAAGGG-
• BamHI -G GATCC-
-CCTAG G-
• BglII -A
GATCT-
-TCTAG
A-
DNA. Tetr
have: Tetr
– An origin of replication.
– A selectable marker (antibiotic
resistance gene, such as ampr and Newer cloning vector
tetr).
LacZ
– Multiple cloning site (MCS) (site MCS
Ampr
DNA cloning requires restriction
endonuclease and DNA ligase
Consider a plasmid with a unique EcoRI site:
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Key features of DNA replication
are used in DNA sequencing
normal
dNTP
Dideoxy NTPs block DNA synthesis
H
ddNTPs block formation of the next
phosphodiester bond during DNA synthesis
A 3´-OH on the last ribose is needed for DNA synthesis
ddNTP
H H
A nucleotide-
specific stop in
DNA synthesis
A mixture of dNTPs and ddNTPs are
used in DNA sequencing
Polyacrylamide gel electrophoresis is used to
visualize the results of the sequencing reaction
Automated DNA sequencing with
fluorescent dyes coupled to each reaction
NCBI.nlm.nih.gov/genome/guide/human/index
Technology now exists to sequence everyone’s DNA
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Understanding the arrangement of genes
may help understand disease
Southern blot: One way to detect genome
structure and disease markers in genomic DNA
-Purify genomic DNA
-Digest with restriction enzyme
-Run agarose gel
Restriction fragment length polymorphisms
(RFLPs) can be associated with disease alleles
Southern Blot
Consider two alleles of a gene.
Allele A has 3 BamHI sites, while
allele a has only two BamHI sites.
probe
HpaI Digest
Nor- Variants
mal 1 2 3
70% of carriers of the sickle cell
gene have a 13.0 kb HpaI fragment.
30% of carriers have 7.0 kb HpaI
fragment
Direct Detection of a Sickle Cell
Mutation by RFLP
A specific hemoglobin mutation
Wild Type Mutant
Pro Glu Pro Val
CCT GAG CCT GTG [DdeI cuts at CTNAG]
DdeI site 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
Polymerase Chain Reaction (PCR)
• Allows quick identification of genetic markers:
Identify bacteria in infections
Identify viruses in virus infections
Paternity testing, genetic counseling, forensics
Can exclude individuals, but cannot prove guilt.
• Requires only small amounts of DNA.
• A repetitive DNA synthesis reaction.
• Thermostable DNA polymerase:
Isolated from bacteria in hot springs or near thermal vents
in the deep ocean.
• Requires gene-specific DNA primers and
deoxyribonucleotide triphosphates (dNTPs).
Polymerase Chain Reaction (PCR)
The major legal problems with PCR are the potential for cross-
contamination between samples and the complexity of explaining what
the results mean to the jury.
PCR can exclude suspects
but cannot prove guilt
• 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.
• Sometimes seemingly strong DNA evidence does not lead to a conviction
(see O.J. Simpson trial).
• 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.
Variable Number of Tandem Repeat (VNTR)
analysis is commonly used in forensics
• Background
• DNA cloning
• DNA sequencing
• Detection of disease genes
• Polymerase chain reaction (PCR)
– PCR basics
– PCR in medicine
– PCR in forensics
Questions?