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Introduction to Cloning and Recombinant DNA Technology. David Bedwell, Ph.D. Department of Microbiology Office telephone: 934-6593 Email: [email protected] The Powerpoint slides for this lecture are available for download at: http://www.microbio.uab.edu/bedwell/index4.html

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Introduction to cloning and recombinant dna technology
Introduction to Cloning and Recombinant DNA Technology

David Bedwell, Ph.D.

Department of Microbiology

Office telephone: 934-6593

Email: [email protected]

The Powerpoint slides for

this lecture are available for download at:

http://www.microbio.uab.edu/bedwell/index4.html

Reference: Molecular Biology of the Cell, 5th Edition, by Alberts et al., published by Garland Science, 2008.


Introduction to cloning and recombinant technology lecture outline
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


Introduction to cloning and recombinant technology lecture outline1
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 Outline

DNA is the genetic material of most organisms (from bacteria to humans)

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 bacteria’s chromosome or its extrachromosomal genetic elements.



Deoxyribonucleic acid dna is a long double stranded chain of nucleotides
Deoxyribonucleic acid (DNA) is a long double-stranded chain of nucleotides

  • DNA is the hereditary material passed on from generation to generation.

  • DNA is made up of four nucleotides: A, C, G, and T.

  • A always pairs with T.

  • C always pairs with G.

  • The two strands of DNA are in an antiparallel configuration.

  • Two complementary DNA strands will separate when heated, and will spontaneously pair together again (hybridize) when cooled.


Dna double helix
DNA Double Helix of nucleotides


Introduction to cloning and recombinant technology lecture outline2
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


What does it mean to clone
What Does It Mean: “To Clone”? Outline

Clone: a collection of molecules or cells, all identical to an original molecule or cell

  • To "clone a gene" is to make many copies of it - for example, by replicating it in a culture of bacteria.

  • Cloned gene can be a normal copy of a gene (= “wild type”).

  • Cloned gene can be an altered version of a gene (= “mutant”).

  • Recombinant DNA technology makes manipulating genes possible.


Restriction enzymes
Restriction Enzymes Outline

  • 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”.


Basics of type ii restriction enzymes
Basics of type II Restriction Enzymes Outline

  • 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
Recognition/Cleavage Sites of Type II Restriction Enzymes Outline

Examples of Palindromes:

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

Cuts usually occurs at

a palindromic sequence

SmaI: produces blunt ends

5´ CCCGGG 3´

3´ GGGCCC 5´

EcoRI: produces sticky ends

5´ GAATTC 3´

3´ CTTAAG 5´


Type ii restriction enzyme nomenclature
Type II restriction enzyme nomenclature Outline

Why the funny names?

  • EcoRI – Escherichia coli strain R, 1st enzyme

  • BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme

  • DpnI – Diplococcus pneumoniae, 1st enzyme

  • HindIII – Haemophilus influenzae, strain D, 3rd enzyme

  • BglII – Bacillus globigii, 2nd enzyme

  • PstI – Providencia stuartii 164, 1st enzyme

  • Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme

  • KpnI – Klebsiella pneumoniae, 1st enzyme


Results of type ii digestion
Results of Type II Digestion Outline

  • Enzymes with staggered cuts  complementary ends

  • HindIII - leaves 5´ overhangs (“sticky”)

    5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’

    3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’

  • KpnI leaves 3´ overhangs (“sticky”)

    5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’

    3’--CCATGG-- 5’ 3’ –C CATGG-- 5’


Results of type ii digestion1
Results of Type II Digestion Outline

  • Enzymes that cut at same position on both strands leave “blunt” ends

  • SmaI

    5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’

    3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’


Restriction endonucleases cleave dna at specific dna sequences
Restriction Endonucleases Cleave DNA Outlineat specific DNA sequences


Dna ligase joins dna fragments together
DNA Ligase joins DNA fragments together Outline

  • Enzymes that cut with staggered cuts result in complementary ends that can be ligated together.

  • HindIII - leaves 5’ overhangs (“sticky”)

    5’ --A AGCTT--3’ 5’ --AAGCTT-- 3’

    3’ --TTCGA A--5’ 3’ --TTCGAA-- 5’

  • Sticky ends that are complementary (from digests with the same or different enzymes) can be ligated together.

  • Sticky ends that are not complementary cannot be ligated together.


DNA Ligase can also join blunt ends Outline

DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme.

  • SmaI -CCC GGG-

  • DraI -AAA TTT-

  • Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT) can be re-cut by SmaI or DraI.

  • Mixed ligation products (CCCTTT + AAAGGG) cannot be re-cut by SmaI or DraI.

-CCCGGG-

-AAATTT-

-CCCTTT-

-AAAGGG-


Any complementary ends can be ligated
Any Complementary Ends Can be Ligated Outline

  • BamHI -G GATCC-

    -CCTAG G-

  • BglII -A GATCT-

    -TCTAG A-

  • Result -GGATCT-

    -CCTAGA-

No longer palindromic, so not cut by BamHI or BglII


Plasmids vehicles for cloning

Amp Outliner

Ori

pBR3224361bp

Tetr

LacZ

MCS

pUC18

Ori

Ampr

Plasmids – vehicles for cloning

  • Plasmids are naturally occurring extrachromosomal DNA molecules.

  • Plasmids are circular, double-stranded DNA.

  • Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.

  • Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.

  • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.


Cloning vectors

Amp Outliner

Ori

pBR3224361bp

Tetr

LacZ

MCS

pUC18

Ori

Ampr

Cloning Vectors

Older cloning vector

  • A cloning vector is a plasmid that can be modified to carry new genes.

  • Plasmids useful as cloning vectors must have:

    • An origin of replication.

    • A selectable marker (antibiotic resistance gene, such as ampr and tetr).

    • Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).

    • Easy to purify away from host DNA.

Newer cloning vector


Chimeric plasmids

CFTR Outline

LacZ

MCS

pUC18-hCFTR

Ori

Ampr

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.


DNA cloning requires restriction endonuclease and DNA ligase Outline

  • Consider a plasmid with a unique EcoRI site:

  • 5' NNNNGAATTCNNNN 3'

  • 3’ NNNNCTTAAGNNNN 5'

  • An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by:

  • a) cutting the plasmid at this site with EcoRI,

  • b) annealing the linearized plasmid with the EcoRI foreign DNA fragment, and,

  • c) sealing the nicks with DNA ligase.

  • 5' NNNNGAATTCNNNN 3'

  • 3' NNNNCTTAAGNNNN 5’

  • This results in a recombinant DNA molecule.


Introduction to cloning and recombinant technology lecture outline3
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


Key features of dna replication are used in dna sequencing
Key features of DNA replication are used in DNA sequencing Outline

  • DNA synthesis occurs in the 5´ to 3´ direction.

  • DNA synthesis requires a template and a primer.

  • DNA replication is semi-conservative (one strand copied).

  • DNA replication is carried out by an enzyme called DNA polymerase.


DNA synthesis requires a 3´-OH to make the next phosphodiester bond during DNA synthesis

normal dNTP


Dideoxy ntps block dna synthesis
Dideoxy NTPs block DNA synthesis phosphodiester bond during 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




Automated dna sequencing with fluorescent dyes coupled to each reaction
Automated DNA sequencing with fluorescent dyes coupled to each reaction

Fluorescent dye coupled to reaction allows visualization of di-deoxy termination events by means of a laser that detects the colored product.

This shows four different reactions as done with the old manual sequencing.


Automated dna sequencing output 4 reactions carried out in one tube
Automated DNA sequencing output- each reaction4 reactions carried out in one tube


Molecular medicine the human genome project
Molecular Medicine: The Human Genome Project each reaction

3.2x109 nucleotide pairs

NCBI.nlm.nih.gov/genome/guide/human/index


Technology now exists to sequence everyone s dna
Technology now exists to sequence everyone’s DNA each reaction

Took just 4 months,

$1.5 million to obtain the entire DNA sequence of James Watson.




Introduction to cloning and recombinant technology lecture outline4
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



Southern blot one way to detect genome structure and disease markers in genomic dna
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
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

Variants

1 2 3

Nor-

mal

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
Direct Detection of a Sickle Cell Mutation by RFLP associated with disease alleles

A specific hemoglobin mutation

Wild TypeMutant

Pro Glu Pro Val

CCT GAG CCT GTG

DdeI site no DdeI site

[DdeI cuts at CTNAG]

AS

AS

SS

AA

Gene encoding sickle cell b-subunit

Gene encoding Wild type b-subunit


Introduction to cloning and recombinant technology lecture outline5
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


Polymerase chain reaction pcr
Polymerase Chain Reaction (PCR) Outline

  • 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) Outline

A thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions.


The first round of pcr
The first round of PCR Outline

94°C

37-65°C

70-75°C



A typical PCR protocol Outline

  • Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.

  • Next, denature the DNA to single strands at 94˚C.

  • Rapidly cool the DNA (37-65˚C) and anneal primers to complementary single strand sequences flanking the target DNA.

  • Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus.

  • Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA.

  • Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.

  • Cool to 4˚C and store or use amplified PCR product for analysis.


Pcr cycle 28 1 billion strands

Outline

5’

A

5’

A

B

A

B

B

PCR cycle 28 – ~1 billion strands

2 original strands.

28 strands starting with primer A, but with no end.

28 strands starting with primer B, but with no end.

~500,000,000 strands starting with primers A (5´) and ending with primer B (referred to as unit-length strand in previous figure).

~500,000,000 strands starting with primer B (5´) and ending with primer A (referred to as unit-length strand in previous figure).


Pcr in medicine
PCR in Medicine Outline

  • Since 1987, PCR has had a major impact on prenatal diagnosis of single gene disorders.

  • Also very important in carrier testing for genetic diseases.

  • Improved speed, accuracy and technical flexibility over previous methods.


Pcr and prenatal diagnosis
PCR and prenatal diagnosis Outline

  • For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid.

  • Single base changes then detected by one or more of following:

    -dot blot (spot hybridization) with oligonucleotides specific for known mutation.

    -restriction enzyme analysis (RFLP).

    -direct sequencing of DNA.

  • Important to be certain of result so combination of two methods provides confirmation.

  • Many other conditions can be detected with same approach, including:

    -Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD).


Pcr to detect hiv
PCR to detect HIV Outline

  • PCR allows the direct detection of HIV genomes in patient blood before the appearance of HIV antibodies.

  • viral DNA/RNA only represents a minute proportion of total cell DNA.

  • Only a small fraction of blood cells are infected (1/10,000).

  • also require high degree of specificity while targeting conserved regions ofDNA to guard against high level of genetic variability characteristic of retroviruses.

  • High risk of cross-contaminating sample with small amounts of amplified DNA from previous sample requires extra precautions to prevent false-positives.

  • PCR can detect 10-20 copies of viral DNA from 150,000 human cells.


Pcr can be more rapid and accurate than other diagnostic tests
PCR can be more rapid and accurate than other diagnostic tests

  • Diagnosis of the middle ear infection known as otitis media. The technique has detected bacterial DNA in children's middle ear fluid, signaling an active infection even when culture methods failed to detect it.

  • Lyme disease, the painful joint inflammation caused by bacteria transmitted by tick bites, can be diagnosed by detecting the disease organism's DNA contained in joint fluid.

  • PCR is the most sensitive and specific test for Helicobacter pylori, the disease organism now known to cause almost all stomach ulcers.

  • PCR can detect three different sexually transmitted disease organisms on a single swab (herpes, papillomaviruses, and chlamydia).


Pcr in forensics
PCR in Forensics tests

  • 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, tissuefrom 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 cross-contamination between samples and the complexity of explaining what the results mean to the jury.


Pcr can exclude suspects but cannot prove guilt
PCR can exclude suspects but cannot prove guilt tests

  • 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
Variable Number of Tandem Repeat (VNTR) analysis is commonly used in forensics

VNTR is based on hypervariable microsatellite sequence polymorphisms within the human genome. These sequences (e.g., CACACA …) are found in many locations in the human genome and vary greatly from person to person.


Using vntr to compare forensic and suspect samples
Using VNTR to compare forensic and suspect samples used in forensics

Individuals A & C are excluded by this analysis. The samples from individual B will be subjected to further tests.


Conclusions
Conclusions 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?


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