Nucleic Acid Manipulation and DNA Cloning

Manipulation of DNA Continued

Nucleic Acid Sequences and Expressed Genes

  • Nucleotide sequence

    • Can deduce protein sequence → structure and function of proteins

    • Can be duplicated, modified, expressed → helps us study proteins

  • Expressed sequence – gene is transcribed and translated

Genes Can Be Isolated by DNA Cloning

  • Clone = an identical copy from single ancestor

  • DNA cloning = isolation, amplification, and modification of a particular gene or DNA segment so that its genetic information may be studied and utilized

  • Recombinant DNA technology or genetic engineering = the methods used to accomplish DNA cloning and related tasks (e.g. purifying DNA)

    • Provides nucleic acids and proteins for other studies and provides a means for studying genes expression under controlled conditions

The Process of DNA Cloning

  • Five general steps:

    • Obtain the DNA fragment to be cloned – by restriction enzyme, PCR, or chemical synthesis

    • Select a molecule of DNA capable of autonomous replication

    • Join the two DNA fragments covalently

      • Gene of interest is paste into a vector, such as a plasmid, to form recombinant DNA

    • Move the recombinant DNA from the test tube to the host organism

    • Select or identify host cells that contain the recombinant DNA

      • Check for their biological properties.

    • Clone – collection of cells that have your DNA of interest

Cloning Vectors Allow Amplification of Inserted DNA Segments

  • Three popular cloning vectors:

    • plasmids – circular DNA (≤10 Kb DNA insert)

    • bacterial artificial chromosomes

    • yeast artificial chromosomes

The Constructed E. coli Plasmid pBR322

  • Key features:

    • Circular

    • Origin of replication (ori) = sequence where replication is initiated

    • Two sets of Antibiotic resistance genes

      • Ampicillin & Tetracycline

      • Used as selectable markers

    • Insertional inactivation

    • Recognition sequences for restriction endonucleases

    • Physically separate from the most chromosomal DNA and replicate independently

    • Can be induced to make 2000-3000 copies

    • Antibiotic kills bact

    • E. coli cells w this plasmid can survive in growth media w Amp + Tet

Restriction Endonucleases

  • Cleave DNA phosphodiester bonds at specific sequences

  • Common in bacteria

    • Eliminates infectious viral DNA

  • Some make staggered cuts.

    • Sticky ends

  • Some make straight cuts.

    • Blunt ends

Restriction Endonucleases Cleave at Specific Recognition Sites

  • Table of Restriction Enzymes and their Recognition Sequences:

    • AluI: AGICT (Arthrobacter luteus)

    • BamHI: G↓GATCC (Bacillus amyloliquefaciens H)

    • BglII: A↓GATCT (Bacillus globigii)

    • EcoRI: G↓AATTC (Escherichia coli RY13)

    • EcoRII: CC(A/T)GG (Escherichia coli R245)

    • EcoRV: GAT↓ATC (Escherichia coli J62 pLG74)

    • HaeII: RGCGC↓Y (Haemophilus aegyptius)

    • HaeIII: GG↓CC (Haemophilus aegyptius)

    • HindIII: A↓AGCTT (Haemophilus influenzae Rd)

    • HpaII: C↓CGG (Haemophilus parainfluenzae)

    • MspI: C↓CGG (Moraxella species)

    • PstI: CTGCA↓G (Providencia stuartii 164)

    • PvuII: CAG↓CTG (Proteus vulgaris)

    • SalI: G↓TCGAC (Streptomyces albus G)

    • TaqI: T↓CGA (Thermus aquaticus)

    • XhoI: C↓TCGAG (Xanthomonas holcicola)

Restriction Sites are Palindromic Sequences with 2-Fold Symmetry

  • Palindromic sequences:

    • Read the same forward and backward (i.e. GAATTC)

    • Cleave two strands of DNA

DNA Ligase

  • “Molecular Glue”

  • Joins two DNA segments with complementary strands by catalyzing the formation of a phosphodiester bond

  • Plays a role in repairing single- or double-strand breaks in duplex DNA in living organisms

  • Covalent bond

  • Cleaved with the same restriction enzyme

Recombinant DNA Construction

  • Recombinant DNA containing your gene of interest, which will be inserted into an E. coli cell.

DNA Ligation Process

  • Restriction enzyme recognition sequence

    • 5'-GAATTC-3'

    • 3'-CTTAAG-5'

  • Restriction enzyme cuts the DNA

    • 5'-G CTTAA-3'

    • 3'-AATTC G-5'

  • Addition of a DNA fragment from another source; fragments stick together by base pairing

  • DNA fragment produced by the same restriction enzyme

    • 5'-GAATTC-3'

    • 3'-CTTAAG-5'

  • DNA ligase seals the strands

  • Recombinant DNA molecule

    • One possible combination

Importance of Using Two Different Restriction Endonucleases in DNA Recombination

  • Question: Why are two different restriction endonucleases used in DNA recombination?

    • A. This is to permit proper orientation of the DNA insert into the plasmid.

    • B. Two different restriction endonucleases are needed to cut both phosphodiester bonds and hydrogen bonds.

    • C. Each is specific for a sequence found on one of the two strands of DNA.

    • D. One is required to produce the open plasmid, and the other produces the insert.

  • Answer: A

    • Using two different restriction endonucleases prevents recircularization of the plasmid and permits proper orientation of the DNA insert into the plasmid.

Selection Marker: β-Galactosidase

  • Bgal

  • How do you know DNA insertion cloning worked?

  • Your DNA of Interest inserted here disrupt lack

  • Not tune pgal

  • Colonies colorless

  • Lact gene encodes func Bgal

  • A insertion cloning

Antibiotic Resistance as Selection Marker

  • Question: A researcher ligates a DNA fragment to pBR322 plasmids (which contain both AmpR and TetR) that were cleaved in the AmpR gene by restriction enzyme, Pstl. Following ligation and E. coli transformation, colonies with recombinant plasmids will grow on which plates?

    • A. tetracycline plates only

    • B. ampicillin plates only

    • C. both tetracycline and ampicillin plates

    • D. neither tetracycline nor ampicillin plates

  • Answer: A. tetracycline plates only

    • Cells that grow on tetracycline but not on tetracycline + ampicillin contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA.

    • Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates.

Electrophoresis Separates Nucleic Acids by Size

  • Ethidium Intercalation

  • Agarose gel electrophoresis

    • DNA travels by size

    • Smaller move faster

Polymerase Chain Reaction (PCR)

  • Technique for generating large quantities of a specified DNA

  • Cell free amplification technique for synthesizing multiple identical copies of any DNA of interest

  • Steps:

    1. Double-stranded DNA of interest is denatured to separate into two individual strands (Denaturation)

    2. Each strand is allowed to hybridize with a primer (Annealing = Rehybridization)

    3. The primer-template duplex is used for DNA synthesis by DNA polymerase (Extension of the new DNA strand)

    4. Denaturation, Annealing, and Extension are repeated to generate multiple forms of target DNA.

DNA Amplification by Polymerase Chain Reaction (PCR)

  • Heat, then cool

  • Use heat stable DNA polymerase (e.g. Taq polymerase)

PCR Primer Considerations

  • Primer Melting Temperature (TmT_m):

    • The temperature at which the oligonucleotide primers are 50% annealed to its exact complement.

    • “The temperature at which primers fall off from the DNA.”

    • Recommended TmT_m of PCR primers: in the range of 55°C to 70°C and within 5°C of each other.

  • Annealing Temp (TaT_a):

    • Based on length and composition of the two primers.

    • Optimal: 5°C below the lowest TmT_m of the primer

    • Too high? - Increased thermal E. No stay bound

    • Too low? - Both primers won't about will anneal not specifically a weak interns are stable low temp

  • Calculation:

    • Longer primer base on Types

    • G/C: add 4

    • A/T: each add 2

  • Tm=4(G+C)+2(A+T)T_m = 4(G+C) + 2(A+T)

  • Stacking use

  • 55°C TaT_a + 0.3-0.4(°C/%GC)

  • TaT_a should be ≤5°C apart

Visual Representation of PCR Amplification

  • Denaturation: Heat to separate strands

  • Annealing: Cool to allow primers to bind

  • Extension: DNA polymerase extends primers

  • Cycle repeats

  • 5′→3′5'→3'

  • 3′→5′3'→5'

  • dNTPs

Design Two PCR Primers

  1. ) DNA template in the PCR reaction test tube is a double stranded molecule. The targeted sequence of nucleotides for this illustration is shown. The 5′ and 3′ orientation of both strands is shown.

  2. ) Denaturation: The hydrogen bonds holding the strands of the double stranded template together are broken by heating the test tube.

  3. ) Annealing: The second step of PCR is to cool the test tube which allows the forward and reverse primer to bind to the template. Find Forward and Reverse Primers with correct ends! (each 6 nts)

Criteria for Primer Design

  1. ) Annealing: The second step of PCR is to cool the test tube which allows the forward and reverse primer to bind to the template.

  • Two most important criteria for primer design

    1. One primer must have a sequence that complements one of the template strands and the other primer must be complementary to the other strand. BOTH strands need to be primed for the replication process.

    2. The primers must bind so that their 3′ ends are ‘pointing’ in the direction of the other primer. This ensures that the sequence between the primers is replicated in the PCR cycles.

  1. ) Extension: final PCR step is when the DNA polymerase enzyme reads the template and connects new nucleotides to the primer’s 3’ end, extending a new complementary strand of DNA. It is heated to around 75°C, optimizing DNA polymerase III activity.

  2. ) The denaturation, annealing, and extension steps are repeated.

PCR Primer Design Considerations

  • In-Lecture Problem 5. PCR has completely revolutionized DNA manipulation, but it is important to design primers that have similar melting temperatures.

    • a. Imagine a case where you are trying to amplify a 1 kB sequence, but you are given primers that differ in Tm by 20°.

      1. ) What, if any, band(s) would you likely see on the gel if you ran a PCR experiment using the Tm of the lower Tm primer as your annealing temperature? Briefly, why?

      2. ) What, if any, band(s) would you likely see on the gel if you ran a PCR experiment using the Tm of the higher Tm primer as your annealing temperature? Briefly, why?

  • Imagine Melting C

  • Cms 50 c

  • TaT_a should 5 C below soc

  • Higher

  • lower

  • Tm vs Tms should be v5 c apart

  • b. You wish to design better primers for this sequence. Examining the sequence, you find that the 5’ end of the sequence is G/C rich and the 3’ end is A/T rich. If you wish to design primers that have similar Tm values, which primer will be longer (the 5’ or 3’ primer)? Briefly, why?

Site-Directed Mutagenesis

  • Alter gene’s nucleotide sequence to alter protein sequence

  • Allows predictions about structural and functional roles of amino acid in protein to be tested in the lab

  • Requires primers