Techniques in Cloning

Polymerase Chain Reaction

• Rapidly creates multiple copies of a segment of DNA

• Uses repeated cycles of DNA synthesis in vitro• Used in DNA fingerprinting, kinship analysis,

genetic testing for mutations, and infectious disease for diagnosis

PCR

Round 0 = 1 copy

Round 35 = billions of copies

PCR players• DNA template – targeted piece of DNA• Primers – small segments of DNA that bind

complementary upstream and downstream of the target on the template

• Taq DNA polymerase – isolated from the Thermus aquaticus bacteria found in hotsprings of Yellowstone Park

• DNA nucleotides in the form of deoxynucleoside triphosphates (dNTPs)

• Reaction Buffer – maintains pH for enzymes

General PCR Process

• Denaturation – split apart the two DNA strands by heating them to 95oC for 30s -1 min

• Annealing – primers bind to target sequence by cooling reaction to 40-60oC for 30s - 1 min

• Extension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 30s - 1 min

Primers• Required for both sides of the target sequence

(forward & reverse primer)• Length of primer is generally 18-30 nucleotides• G/C content and intra-complementarity are a

concern when designing primers• Actually not a single primer for each but a mixture of

primers (oligoprimers) if the sequence of the target is not known

• If amino acid sequence of gene product is used then degenerate primers must be used

• Initial forward primer is GABTATGTTGTTGARTCTTCWGG B=G/T/C R=G/A (purines) W =A/T

Nested PCR

• Initial PCR primers are degenerate and based on a consensus sequence

• The chances that the initial primers will bind to sequences other than the target are high

• A second set of primers designed to be more specific to target is used

• They are nested within the initial primers and are not degenerate thus much more specific to the target gene

Nested PCR

Our experimentTube setup: Add the following to a P3 tube (with PCR reaction pellet)

5 ul Target DNA template (P1)10 ul Primer set (P2)15 ul Enzyme -grade water (P4)

PCR Plan

Initial Denaturation 94oC for 5 minutes

Then 30 Cycles of:

Denaturation 94oC for 30 sec

Annealing 50oC for 30 sec

Extention 72oC for 30 sec

Final Extension 72oC for 5 minutes

Hold 15oC forever

Gel Electrophoresis

• Definition: the process of separating molecules based on size and charge

• Agarose: highly purified agar, heated and dissolved in buffer. Forms a matrix of pores for molecules to travel through.– Smaller molecules travel further– Molecules migrate towards the positive (red) end

of the chamber

Gel Electrophoresis

• Process– Make Agarose gel

• Thinner gels (0.8%) yield better results for larger DNA

– Prepare samples• Restriction enzymes used to cleave at specified sites

– Apply samples to gels, apply current• If samples run from positive end they will run off the gel

– Stain gels to see bands• Would not be able to see bands if we did not stain

Gel Electrophoresis• DNA molecules have a negative charge

– This allows them to migrate towards the positive end of the chamber

• The samples and the electrophoresis chamber use specialized buffers. Using TAE/TBE buffer helps stabilize the sample and allows the reaction to occur quicker in the chamber.– If water were in the chamber instead of TAE/TBE buffer

the reaction would take much longer or migration may not occur at all

• Stains: ethidium bromide will cause the bands to glow orange under UV light. Fast stain will result in blue bands

Uses for Gel Electrophoresis

• DNA fingerprinting or profiling– Paternity testing– Crime scene sample analysis– Identification of bacteria and other pathogens

• Who is credited with discovering the DNA profiling process?– Alec Jefferies in 1985

Gel Electrophoresis

PCR purification

• Small impurities can have a negative effect on the ligation of the PCR product to vector DNA

• Impurities include unincorporated dNTPs, polymerases, primers and small primer-dimers.

• A PCR spin column will remove the impurities in less than 4 min.

Restriction enzymes (endonucleases)

• Definition: class of enzymes that cleave (cut) DNA at a specific and unique internal location along its length.– Makes 2 incisions, one through each of the sugar-phoshate

backbones of the double helix• They can be naturally produced in bacteria and the

bacteria use them as a defense mechanism against viral infection– The enzymes chop up the viral nucleic acids and destroy

the virus– More than 3,000 known restriction enzymes– Common ones are: EcoRI, Psti, HindII

Restriction enzymes (endonucleases)

• Discovered in late 1970s by Arber, Smith and Nathans

• The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases

• Uses:– To insert new segment of DNA– To cut specific segments of DNA to study– To cut segment from one gene to insert it into another

• Genetic engineering or recombinant DNA• Need suitable host, vector for carrying plasmid, way to get host to

take up gene

Restriction Digests

• Each enzyme cuts DNA at a specific sequence= restriction site

• Many of the restriction sites are 4 or 6-base palindrome sequences

Enzyme cuts

Fragment 1 Fragment 2

Enzyme Examples

EcoRI G-A-A-T-T-C

C-T-T-A-A-G

HindIII A-A-G-C-T-T

T-T-C-G-A-A

BamHI G-G-A-T-C-C

C-C-T-A-G-G

Bgl II A-G-A-T-C-T

T-C-T-A-G-A

Restriction enzymes (endonucleases)

• Sticky ends: when unpaired length of bases (5’ AATT 3’) encounter an unpaired length of sequences (3’ TTAA 5’), they will bind or are “sticky” for each other.

• Blunt ends: same length sequences or DNA section cut in half– Joining of two blunt ends is ligation

Restriction Digest

• Restriction Buffer provides optimal conditions:– NaCl provides correct ionic strength– Tris-HCl provides proper pH– Mg+2 is an enzyme co-factor

• Body temperature (37oC) is optimal– Too hot kills enzyme– Too cool takes longer digestion time– Specific enzymes have specifc temps and times

Ligation

• T4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ phosphate on another piece.

• Requires ATP and Mg+2

• Insert to vector DNA ratio should be 1:1• Proofing reading DNA polymerase removes

dangling 3’A of PCR product

Products of Ligation

• Self-ligation of vector• Ligation of vector to primer-dimers• Ligation of multiple inserts• Self-ligation of inserts• Ligation of one insert into vector

Bacterial DNA

Plasmid DNA

Bacterial cell

Genomic DNA

Plasmids are good vectors:

• small (2,000 – 10,000 bp) • circular, self-replicating • high copy number• multiple cloning sites (MCS)• selectable markers (Amp-resistance)• screening (reporter genes, positive select)• control mechanisms (lac operon)• can handle the size of the insert

Transformation

• Once PCR product (insert) has been ligated into a plasmid, the plasmid be introduced into a living bacterial cell to replicate.

• Two methods of transformation:– Electroporation– Heat Shock

• Both methods make cells competent - able to take up plasmids

Transformation Steps

• Wash away growth media from cells• Place cells in ice cold calcium chloride which most

likely hardens the cell membrane• Add plasmid to cells• Move cells to hot environment (usually 42oC) causes

membrane pores to open so plasmid can enter• Add nutrient media to cells to allow them to recover

from stress• Plate cells on selective growth plates (Amp and IPTG

(increases expression of ampr gene)

Microbial Culturing

• Pick a colony from the transformed cells to innoculate a liquid culture

• Liquid culture (broth) must have selective antibiotic (Amp) in it.

• Choose a single colony from the plate• Under favorable conditions, a single bacteria

divides every 20 minutes and will multiply into billions in 24 hours

Plasmid Purification

• To confirm that the engineered cells have been transformed with the correct DNA

• Different methods– Lysozyme Method – Alkaline Cell Lysis Method– Column Methods (Aurum, EZNA)

Plasmid preps

• Spectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/ml

• Column can process up to 12 OD●ml of bacterial host cells

• Cells disrupted with a lysis buffer• DNA binds to membrane of column, is washed

and then eluted with aqueous buffer.

Restriction Digest

• Restriction Buffer provides optimal conditions:– NaCl provides correct ionic strength– Tris-HCl provides proper pH– Mg+2 is an enzyme co-factor

• Body temperature (37oC) is optimal– Too hot kills enzyme– Too cool takes longer digestion time

Lambda DNA• Lambda DNA

comes from a bacteriophage

• Genomic DNA of Lambda is well studied and used in research as a size markers for DNA pieces

• Arrow mark HindIII restriction sites

DNA Sequencing

• Determining the exact order of the nucleotide sequence in a DNA molecule.

• Use to take days, now takes hours• Have sequences of entire genones for over

700 organisms

Sanger Method• Prepare single-stranded DNA template to be

sequenced• Divide DNA into four test tubes• Add primer to each tube to start DNA synthesis• Add DNA polymerase• Add labeled deoxynucleotides (dNTP) in excess.

Labeled with radioactive or fluorescent tags• Add a single type of dideoxynucleotides (ddNTPs) to

each tube. When incorporated in sythesized strand, synthesis terminates.

• Allow DNA synthesis to proceed in each tube• Run newly synthesized DNA on a polyacrylamide gel

Reading the Sequence•In the tube with the ddTTP, every time it is time to

add a T to the new strand, some Ts will be dTTP and some will be ddTTP.

•When the ddTTP is added, then extension stops and you have a DNA fragment of a particular length.

•The T tube will, therefore, have a series of DNA fragments that each terminate with a ddTTP.

•Thus the T tube will show you everywhere there is a T on the gel

•Same thing happens in all tubes

•Read gel from top to bottom looking at all four lanes to get the sequence.

Automated Sequencing

• Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye.

• Now reaction can be run in one tube• Use capillary electrophoresis rather than the

standard polyacrylamide slab gel.• When DNA fragment exits gel, the dyes are

excited by a laser and emit a light that can be detected .

• Produces a graph called a chromatogram or electopherogram

Automated Sequencing

Bioinformatics

• Computerized databases to store, organize, and index the data and for specialized tools to view and analyze biological data

• Uses include– Evolutionary biology– Protein modeling– Genome mapping

• Databases are accessible to the public • Allow us to record, compare, or identify a DNA

sequence

Types of RNA

• Messenger RNA (mRNA)• Tranfer RNA (tRNA)• Ribosomal RNA (rRNA)• Signal Recognition Particle RNA (SRP RNA)• Small Interfering RNA (siRNA) – gene reg• Micro RNA (miRNA) – gene reg.

RNA Interference (RNAi)

• Dicer enzyme cuts dsRNA up into smaller siRNA which then complex into the RNA-induced silencing complex (RISC) which then cuts up the mRNA

• dsRNA can be engineered so that genes can be systematically shut down

Reverse Transcriptase PCR

• Use reverse transcriptase to make a DNA copy of mRNA• Copy called cDNA• Allow scientists to study the level of gene expression in

a cell

Northern Blots

• Run mRNA on a gel• Transfer it to nitrocellulose membrane• Add labeled cDNA probes to

the membrane and hybridize the probes to the RNA

• Allows you see what genes are expressed

DNA Microarray (Chip)

• Adhere genes to chip• Collect mRNA from cells• Make labelled cDNA

from mRNA (red + green)• Add cDNA to chip• Measure signal