Ch. 10 Gene Isolation And Manipulation Pt. 1

Front 1

What is recombinant DNA technology?

Back 1

The collective techniques for obtaining, amplifying, and manipulating specific DNA fragments. Uses in vivo (in a host cell) and in vitro (in a test tube) techniques.

Front 2

What is gene cloning?

Back 2

The process of isolating and making copies of a gene of interest.

Front 3

What components make up the molecular geneticist's toolbox?

Back 3

1. Restriction enzymes (cut DNA)

2. DNA polymerase (synthesize more DNA of interest)

3. DNA nucleotides (dNTPs)

4. DNA ligase (joins DNA fragments together)

5. Cloning Vectors (CVs. Harbor the gene of interest or GOI for cloning)

Front 4

What is amplification?

Back 4

The production of many DNA copies from one master region of DNA.

Front 5

Describe gene cloning in vivo.

Back 5

1. First isolate DNA from organism or chromosome. Cut up this donor DNA with restriction enzyme endonuclease

2. Cut DNA cloning vector to insert (ligate) DNA donor fragments into CV

3. Transform a host cell with the CV-GOI recombinant.

4. Replicate host cell.

Front 6

What are cloning vectors?

Back 6

A plasmid or phage chromosome used to carry a gene of interest to be replicated.

Front 7

What are recombinant DNA molecules?

Back 7

Novel DNA sequences formed by the combination of two non homologous DNA molecules. E.g. A vector with inserted donor DNA.

Front 8

Describe in vitro gene cloning.

Back 8

Polymerase Chain Reaction (PCR)

1. Denature dsDNA to ssDNA

2. Add (anneal) primers to flank GOI

3. Extend primers with DNA polymerase.

4. Rinse and repeat to amplify amount of target DNA sequence.

Front 9

What is PCR?

Back 9

Polymerase Chain Reaction. A type of DNA amplification that uses 2 primers that hybridize to opposite ends of the DNA segment (one for leading and one for lagging strand) and, over successive cycles, prime exponential replication of that segment only.

Front 10

What is the role of an endonuclease in generating recombinant DNA molecules?

Back 10

They are restriction enzymes. Different ones recognize different specific restriction sites (usually palindromes) and cut nucleic acids at that site to prepare for insertion into a cloning vector.

Front 11

What bonds do endonuclease cut?

Back 11

The phosphodiester bonds of the sugar/ phosphate backbone.

Front 12

What is a restriction fragment?

Back 12

A DNA fragment resulting from cutting the DNA with a restriction enzyme. Fragment is determined by restriction sites, and a specific RE site will produce the same fragments every time.

Front 13

What is a symmetrical cut?

Back 13

A cut at a restriction site that produces blunt ends. This means the ends are the same and have no overhanging ends.

Front 14

What are asymmetrical cuts?

Back 14

Cuts by REs that leave overhangs on the ends of the DNA stands. These are called "sticky" ends because they are sections of ssDNA that can more easily recombine/ compliment/ base pair with cloning vectors cut with the same RE as the donor DNA.

Front 15

What 4 elements must E. coli plasmid CVs have?

Back 15

1. Origins recognized by host replication proteins.

2. Multiple cloning sites (also called polylinkers), which is where the GOI is inserted.

3. Selectable Marker, typically an antibiotic resistance gene so hosts that don't uptake the CV die when plated on a gel containing the corresponding antibiotic

4. The lacZ gene (spans the MCS) and codes for the enzyme beta-galactosidase

Front 16

What are the steps for in vivo amplification?

Back 16

1. Cut (digest) the CV and donor DNA with the same restriction enzyme. Preferably one that makes sticky ends.

2. Mix the CV and donor DNA fragments together

3. Add ligase so inserted donor DNA (hopefully containing GOI) and CV sugar/ phosphate backbones will seal

4. Transform competent E. coli with your newly made cloning vector.

5. Plate cells on media containing X-gal and ampicillin

Front 17

What are the three possible outcomes of creating a recombinant cloning vector?

Back 17

1. A recircularized CV with no insert

2. A recombinant CV with the GOI inserted (good)

3. A recombinant CV with no GOI insertion

Front 18

Why would you plate your CV host cells on a medium containing X-Gal and ampicillin?

Back 18

The ampicillin will kill any cells that aren't successfully transformed by a CV.

The restriction enzyme is designed to cut in pokylinker so insert will disrupt the lacZ gene. This prevents the cell's beta-galactosidase from interacting with the X-Gal substrate to produce a blue pigment. This means white colonies have a CV with a donor insert while blue colonies have a CV with no donor insert.

Front 19

What are two artificial ways to make host E. coli cells competent (make membrane more permeable) to take up CVs?

Back 19

Bathe in cold CaCl2 and heat shock

Electroporation

Front 20

What prevents self-ligation of a CV?

Back 20

Alkaline phosphatase

Front 21

What is colony hybridization?

Back 21

The process used to determine which in vivo colonies contain your GOI rather than some other DNA fragment.

Front 22

What are the steps to colony hybridization?

Back 22

1. Create a master plate of host colonies that definitely have a cloning vector.

2. Place a nylon membrane on master plate and lift it off to create a replica containing some of the cells from host colonies.

3. Treat membrane with detergent to lyse cells and fix DNA by UV light.

4. Add sodium hydroxide (NaOH, which has a high pH). This denatures dsDNA into ssDNA.

5. Add a radiolabeled probe (complimentary ssDNA to target gene). This allows the probe to bind to the denatured DNA of interest.

6. Wash off unbound probes and expose membrane to x-ray film

7. Dark spots on film indicate probe hybridized with GOI. Compare X-ray film to master plate to ID GOI colonies

Front 23

What is cDNA?

Back 23

Complementary DNA. It is DNA transcribed from an mRNA template through the action of the enzyme reverse transcriptase and a DNA polymerase.

Front 24

When would you use cDNA? Why?

Back 24

When you're only interested in the coding sequence of your gene of interest. This is because cDNA is made from processed mRNA, so all the introns have been removed.

Front 25

What type of mRNA is used to make cDNA? Why?

Back 25

Eukaryotic. This is because it has a polyA tail so the oligo-dt primer can bind. Prokaryotes don't have a polyA tail, so it won't work.

Front 26

How do you synthesize cDNA?

Back 26

1. Isolate mRNA from cells and add oligo-dT primer (a short sequence of DNA Ts that will complement the mature mRNA's polyA tail)

2. Add reverse transcriptase and dNTPs to extend oligo-dT primer (gives you a DNA complementary copy of the mature mRNA sequence)

3. Add enzyme RNase H to remove/ degrade mRNA to release DNA copy.

4. Add DNA polymerase to make the complementary strand to your ssDNA copy.

Front 27

What are restriction site adaptors for?

Back 27

cDNA molecules have blunt ends. Must add restriction site adaptors to provide them with sticky ends.

Front 28

How do you insert a cDNA into a cloning vector?

Back 28

1. Ligate oligo-nt linkers with restriction site sequence (provides restriction site adaptors to allow for certain of sticky ends).

2. Cut cDNA and CV with same RE

3. Mix the two together to allow insertion into CV

4. Add ligase to seal the insert

Front 29

What process is PCR based on?

Back 29

DNA replication

Front 30

What do you need to perform PCR?

Back 30

1. The target DNA (GOI)

2. 2 primers (one each for leading and lagging strand)

3. A heat resistant DNA polymerase (Taq Polymerase)

4. DNTPs

5. A thermal cycler

Front 31

What is the procedure for PCR?

Back 31

1. Denature GOI DNA at 95 degrees C

2. Aneal commentary primers to GOI at 50-65 degrees C

3. Extend primers at 72 degrees C

4. Repeat 20-40 times.

Target DNA is doubled with each round of PCR, increasing what you have exponentially.

Front 32

What is a polylinker?

Back 32

A short segment of DNA which contains many restriction sites. Used in cloning vector plasmids.

Front 33

How do you know if your target sequence was successfully amplified during PCR?

Back 33

You run the product on a gel using gel electrophoresis. Compare your product to a DNA ladder (markers that contain DNA/RNA fragments of known length). If you know how long your product is, it should match the same length marker.

Front 34

What are the main reasons PCR levels off instead of continuing to make more product?

Back 34

1. Run out of dNTPs

2. Run out of primers

Front 35

How do you insert a PCR product into a cloning vector?

Back 35

The primers must contain restriction sites. Cut DNA and CV with restriction enzyme. Mix together to allow insertion. Ligate to seal backbone.

Front 36

What is RT-PCR?

Back 36

Reverse transcriptase PCR. Used to detect the presence of a specific mRNA.

Front 37

What is the procedure for RT-PCR?

Back 37

1. Make cDNA from mRNA using reverse transcriptase.

2. Amplify product using PCR

3. Run on a gel to determine success

Essentially same as PCR, only using cDNA, so your product is made from mRNA and contains no introns.

Front 38

What does semiquantitative mean in relation to PCR and RT-PCR?

Back 38

It means we can get some idea of the quantity of the product of DNA, but not exactly. Or the quantity of RNA expressed, but not exactly.

Front 39

What is qPCR?

Back 39

Quantitative (real time) PCR. It can detect the quantity of DNA present or RNA expressed.

Front 40

What is the procedure for qPCR?

Back 40

1. Create cDNA via reverse transcriptase.

2. Perform PCR using SYBR green or Taqman probes.

Front 41

How does SYBR green work?

Back 41

It's a fluorescent intercalator that sits between the DNA bases. We can use fluorescent concentration to get a quantitative measurement.

Front 42

How does TaqMan work?

Back 42

They are fluorescent probes that anneal within a DNA region amplified by specific primers. Bind to the outside of the DNA backbone. Greater fluorescence means more DNA product.

Front 43

What is a DNA library, and what are the 2 kinds?

Back 43

It is a collection of recombinant vectors.

1. Genomic: contains fragments from a whole chromosome

2. cDNA: Contains only cDNA (expressed genes)

Front 44

How do you construct a cDNA library?

Back 44

Typically by using specific tissues where the protein product from the target GOI is found in high concentration. Follow the procedure for making and inserting cDNA into a CV and the procedure for transforming bacteria. Or keep as plasmids.

Front 45

How do you construct a genomic library?

Back 45

By using a restriction enzyme to randomly cut chromosomes into fragments and inserting those fragments into cloning vectors. Use the same procedure for inserting DNA into CV and transforming bacteria. Or can keep as plasmids.

Front 46

Why is it important to choose the right vector for your target DNA/DNA library? What are the different vectors?

Back 46

Genes come in various sizes, so need vectors that can hold the desired gene insert.

Plasmids, lambda phages, fosmids, BACs (bacterial artificial chromosomes), and YACs (yeast artificial chromosomes)

Front 47

For plasmids, what is the typical host, average insert size in kb, # of possible clones*, type of insertion, plated results, and type of library used in?

*representative of human genome, 3x10^9bp

Back 47

1. E. coli

2. 4kb

3. 3x10^9bp / 4000bp = 750,000

4. Transformation

5. Bacterial colonies

6. Genomic & cDNA libraries.

Front 48

For lambda phages, what is the host, average insert size in kb, type of insertion, # of clones*, and plated result?

*representative of human genome, 3x10^9bp

Back 48

1. E. coli

2. 15kb

3. Infection

4. 3x10^9bp / 15000bp = 200,000

5. Plaques

6. cDNA or genomic libraries

Front 49

For fosmids, what is the host, average insert size in kb, type of insertion, # of clones*, and plated result? *representative of human genome, 3x10^9bp

Back 49

1. E. coli

2. 40kb

3. Transduction

4. 3x10^9bp / 40000bp = 75,000

5. Bacterial colonies

6. Genomic libraries

Front 50

How do you screen a genomic library for a gene of interest when using plasmid, fosmid, or BAC vectors?

Back 50

Colony hybridization. Same steps as used in identifying if in vivo cells have the GOI in them.

Hint: starts by placing a nylon membrane over a master plate of bacterial colonies.

Plaque hybridization is used to ID GOI in genomic library created using phages.

Front 51

What is a Southern blot? What is the procedure?

Back 51

A process used to detect particular gene sequences within a mixture of chromosomal fragments.

1. Perform gel electrophoresis with one lane containing radiolabeled size markers.

2. Transfer DNA from gel to a membrane.

3. Hybridize membrane with unique nucleic acid probes (radiolabeled and designed to complementary sequence of interest)

4. Wash away unbound probes.

5. Expose membrane to X-ray film. You will see the fragments hybridized with probes. Those fragments will line up with the markers to help ID sequence of interest.

Front 52

What is a Northern blot? What is the procedure? What do different bands mean?

Back 52

A procedure used to detect RNA. If a particular gene of interest is expressed, its mRNA (and any splice variants) can be detected.

Same procedure as Southern blot.

A band in one lane means the gene is expressed. If you see a different band in another lane, then an alternative splice variant of the same gene is expressed.

Front 53

What is RT-PCR?

Back 53

Reverse transcriptase PCR. Can measure RNA expression. Faster and less prone to errors than a Northern blot.

1. RT-PCR

2. Gel electrophoresis

3. Southern blot

Front 54

What is a Western blot? What is the procedure?

Back 54

A procedure used to detect a specific protein from a mixture of proteins. Uses antibodies as probes.

1. Dissolve and isolate proteins in Sodiumdodecyl Sulfate and then boil (helps unfold proteins and coats them with a negative charge for gel electrophoresis)

2. Separate protein by SDS-PAGE (Sodiumdodecyl Sulfate - Polyacrylamide gel electrophoresis). PAGE works better for proteins than agarose.

3. Transfer proteins onto nylon membrane.

4. Add primary antibody that binds to protein of interest. Wash off excess.

5. Add secondary antibody, which binds to primary, and is covalently linked to a report enzyme.

6. Add enzyme substrate linked to secondary antibody to produce color or luminescence.

7. Expose to X-ray film to find target protein.

Front 55

What is DNA sequencing?

Back 55

The process of determining the nucleotide sequence of a DNA fragment using the principle of DNA replication.

Front 56

What is the procedure for DNA sequencing?

Back 56

1. Set up reaction cocktail

- DNA template to be sequenced

- radioactively labeled primer to anneal to template

- DNA polymerase

- dNTPs

2. Divide mixture into four tubes (each one spiked with only one kind of the 4 ddNTP)

3. Start reaction

4. When a ddNTP randomly inserts into the growing strand, polymerization stops

5. Run the four reaction mixtures on a polyacrylamide gel, then expose to film.

6. DNA bands are read from bottom to top = shortest to longest.

Front 57

What is automated DNA sequencing?

Back 57

Same reaction as normal DNA sequencing, but the ddNTPs are labeled with different colored fluorescence. A fluorescent reader detects each tag as the ddNTPs emerge from the gel.

Front 58

What is in situ hybridization?

Back 58

It means "in place." Used to visualize gene activity directly in fixed cells or tissues.

Ex: Fire and Mello in their gene silencing experiment used this method with their C. elegans embryos. A labeled probe complementary to their target mRNA was added. If cells expressed the mRNA protein product, the mRNA in the cell will bind to the probe and become labeled. After washing away unbound probes, can observe under a microscope.

Front 59

If you begin PCR with 8 copies of dsDNA, how many copies would be present after 35 cycles? Why?

Back 59

8x2^35 = 2.7x10^11

If you start with one copy, that's 2 ssDNA strands. The first step of PCR is to denature DNA, so the calculation is 2^n, meaning 2 strands to the power of however many cycles. Since we're starting with 8, we must multiply 2^35 by 8.

Front 60

Can you make cDNA from bacterial mRNA? Why or why not?

Back 60

No. Bacterial mRNA doesn't have a poly A tail, and that is where oligo-dT primers bind.

Front 61

When sequencing a gene, how do you read the gel? What is the orientation? Is the sequence that of the template or non-template strand?

Back 61

Bottom to top (shortest fragment to longest). Starts with 5' end to 3' end. Primer is not included when reading the gel because all the fragments have it. You will have the sequence for the non-template strand. Aka: the template strand's complementary sequence.