Genetics Untested Material: Molecular Analysis and Biotechnology I and II

What are the steps involved with recombinant DNA?

1. Finding the target gene in the source genome

2. Separation of the target from the rest of the genome

3. Stabilization of the target for maintainance in the new host

4. Identification and isolation of host cells that have received the target DNA

May include manipulation of the sequences so that the gene can be expressed in the new host

What is the purpose of cutting and recombining DNA fragments in vitro?

Restriction enzymes from bacteria are used to cut DNA at specific sequences. These cuts often result in “sticky'“ cohesive ends that aid in recombination.

What is added to form phosphodiester bonds between adjacent bases in the sequences that are being joined?

DNA ligase

How many restriction sites are found within a genome?

• By knowing the GC ratio of the target DNA, the chance of a specific base at a particular site can be determined

• Then number of bases in the recognition site factors into the determination of how many sites may be found. Longer recognition sequences will occur less frequently

• The size of the genome will also factor into the estimation of the number of a restriction sites present. Bigger genomes will likely have more sites.

How many BamHI sites are there in the E. coli genome?

• BamHI recognition site: GGATCC

• E. coli GC% ratio over genome: 50.8%

• Likelihood of GGATCC can be determined using the multiplication rule: 0.254 × 0.254 × 0.246 × 0.246 × 0.254 × 0.254 = 0.00025. The E. coli genome is about 4.6 million base pairs long. So, there are approximately 4.6 × 10^6 × 0.00025 or about 1150 BamHI sites.

Why are they called “Restriction” Enzymes?

• They are part of bacterial Restriction/Modification Systems for inhibiting foreign DNA in the cell

• These systems modify the host (self) DNA, typically by methylation of specific bases

• Endonucleases are unable to bind to modified DNA, but recognize un-modified sequences and cleave DNA at those sites

What are some Restriction/Modification systems?

• Restriction endonucleases recognize specific sequences of DNA and cleave it

• Methylases modify DNA at the recognition sequences, preventing the endonucleases from acting. So, the systems are present in various orgaisms that allow for the degradation of foreign DNA, without destroying the self DNA

What are CRISPR-Cas Systems from Bacteria?

• Discovered in bacterial systems as a defense system against invading DNAs (viruses and plasmids)

• Clustered Regularly Interspaced Short Palindromic Repeats are expressed from DNA to produce crRNA. These sequences have acquired DNA from foreign sources, and with a CRISPR-associated (Cas) protein, can recognize and cleave such DNA if it is re-introduced into the bacterial cell

• This has been referred to as bacterial immunity

What is CRISPR-Cas Genome Editing?

• Can be performed in vivo

• The two required RNAs in the native system have been engineered into a single guide RNA (sgRNA) that is able to recognize targeted DNA sequences and cleave there with high specificity

• Repair of the cleaved DNA allows mutation at specific sequences

• These systems have been developed to allow insertion of new DNA at these sites

What is Gel Electrophoresis?

• Gel is formed in a cast. It may be made of polyacrylamide or agarose, depending on the application

• Samples are applied to the wells. Samples contain glycerol so that they sink into the well, and a dye so they can be seen

• An electric current is applied across the gel causing the migration of charged molecules in the sample

• Separation occurs according to size for molecules (e.g nucleic acids) with a constant charge to weight ratio. Visualization of DNA usually requires additional steps such as staining or autoradiography

What are some characterizations of gel electrophoresis?

• Gel electrophoresis can be used to separate DNA molecules on the basis of their size and electrical charge

• Smaller fragments will travel farther during electrophoresis

• Visualization can be carried out by staining the DNA (soaking the gel) in ethidium bromide which intercalates the DNA and fluoresces orange under UV light

What is the detection of sequences via Hybridization?

• The probe is an oligonucleotide (typically synthetic)

• The label may be radioactive, fluorescent, or enzymatic

• Hybrid molecules indicate sequence similarity between probe and the nucleic acid to which it is bound

What are some uses of Nucleic Acid Hybridization?

• Southern Blotting (detecting DNA)

• Northern Blotting (detecting RNA)

• Genetic Disease Diagnosis

• Pathogen Identification

• Microarrays (assaying gene expression)

• Polymerase Chain Reaction

What does Southern Blotting do?

This procedure enables identification of DNA fragments in the original digest that have specific sequences of interest, based on their complementation to the labeled probe

Moving DNA fragments of choice into a new host often involves the use of a ____________

plasmid cloning vector

What are the steps for plasmid cloning vector?

1. First, a cloning vector must contain an origin of replication recognized in the host cell so that it is replicated along with the DNA that it carries

2. 2. Second, it should carry selectable markers—-traits that enable cells containing the vector to be selected or identified

3. Third, a cloning vector needs a single cleavage site for each of one or more restriction enzyme used

What are the three important properties of cloning vectors?

• Origin of replication for maintenance in new host

• Marker (gene) to enable selection of bacteria that contain the vector

• Restriction sites where genes of interest can be inserted

The ____ plasmid is a typical cloning vector

pUC19

The ____ gene is interrupted and inactivated when DNA is cloned into the restriction sites located within the gene

lacZ

The lacZ gene can be used to screen bacteria containing ______

recombinant plasmids

What do expression vectors enable?

Regulated Transcription of cloned genes

What do expression vectors contain?

• Expression vectors contain operon sequences that allow inserted DNA to be transcribed and translated

• They also include sequences that regulate—turn on or turn off—the desired gene

Bacteria are not the only hosts for Recombinant DNA

True

Agrobacterium tumefaciens can transfer______

DNA located between Ti plasmid’s TL and TR flanking sequences into plant cells during infection of the plant

What are the steps used to produce genetically modified tobacco?

1. The gene Bacillus thuringiensis encodes a protein that is toxic to the larvae of hornworms which destroy the leaves of tobacco and the gene cloned into an E. coli plasmid and linked to the neo gene

2. The gene constructs (neo-Bttoxin) were cloned into an expression system that works for eukaryotic hosts

3. A. tumefaciens was transformed with the new plasmids which underwent recombination with the Ti plasmid. The resultant Ti-derived plasmid can transfer the cloned toxin gene along with the neo and the regulatory sequences into the plant host.

4. The neo gene confers resistance to kanamycin for the plant cells that incorporated the cloned gene constructs. Tobacco plants derived from these cells were tested for toxicity to hornworms. Those that were toxic were bred commercially.

What are some characteristics of the Polymerase Chain Reaction (PCR)?

• It amplifies selected DNA sequences

• the polymerase chain reaction is used for amplifying sequences from a small sample of DNA

• PCR can increase the proportion of a particular DNA sequence in a mixed DNA population

What does PCR reaction consist of?

DNA template, Oligonucleotide primers, dATP, dCTP, dGTP, dTTP, heat-stable DNA polymerase

What can PCR also be used for?

A diagnostic tool and to find rare sequences in a DNA sample

What are some Caveats of using PCR vs Cloning to Generate Quantities of DNA for Manipulation?

• Requires prior knowledge of at least part of the sequence so that primers can be made

• Contamination can result in amplification of unexpected DNA species

• Taq DNA polymerase lacks proofreading so fidelity is a consideration. Fidelity has been improved with alternative heat-stable polymerases

• Size limitations may be restrictive dependent upon the sequences to be amplified

What are some sources of DNA for cloning?

• Restriction digests of DNA from various sources (e.g library construction)

• cDNA libraries (libraries are DNA that has been made from messenger DNA)

• PCR products

What are DNA libraries?

• Collections of clones of DNA from a source are called DNA libraries

• They can be screened and serve as a source of DNA sequences of interest

• The DNA clones are carried by cloning vectors, within a (usually) bacterial strain

• Genomic libraries contain clones of all the genomic DNA from a source

• cDNA libraries contain clones of all the expressed DNA from a source

Steps of constructing a Genomic Library:

1. Cleave with restriction nuclease

2. DNA fragments inserted into plasmids using ligase

3. Introduction of plasmids into bacteria

How to construct a cDNA library

1. Isolate mRNA

2. Synthesize complementary strand in DNA

3. Remove RNA

4. Replace with new DNA strand

5. Clone into vector

Genomic and cDNA libraries can be screened with a probe to find the gene of interest

True

How do you localize genes within the chromosome and localize gene expression (RNA) spatially within tissues?

By fluorescent in situ hybridization (FISH)

_______ length polymorphisms are genetic markers that can be used in mapping

Restriction fragment

What are restriction fragment length polymorphism (RFLP) Analysis?

• An Mstll restriction site present in the normal gene is lost with mutation

• CTGAGG —— CTGTGG

Sequencing DNA characteristics

• Base-specific chemical cleavage

• Di-deoxynucleic acid incorporation

- Sanger sequencing using radioactive substrates

- Use of fluorescent markers

- Cycle sequencing

What type of cleavage is Maxim-Gilbert sequencing?

chemical cleavage

What does Sanger (dideoxy) sequencing contain?

Template, primer, dNTPs + one ddNTP, and DNA polymerase in four separaate reactions

Deoxyribonucleoside triphosphate (dNTP) vs Dideoxyribonucleoside triohosphate (ddNTP)

missing hydroxyls at 3 prime

Deoxyribonucleoside triphosphate

• Allows strand extension at 3 prime end

• normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dGTP, and dTTP)

• oligonucleotide primer for DNA polymerase and single-stranded DNA molecule to be sequenced

Dideoxyribonucleoside triphosphate

• Prevents strand extension at 3 prime end

• small amount of one dideoxyribonucleoside triphosphate (ddATP)

• rare incorporation of dideoxyribonucleoside by DNA polymerase blocks further growth of the DNA molecule

Characteristics of sequenccing with fluorescent ddNTPs

• Has template, unlabelled primer, dNTPs, all ddNTP, and DNA polymerase in one reaction

• Completed reaction is passed through a gel filtration column to separate the extension products according to size, and then through a fluorometer to measure the emission spectra

What are some characteristics of DNA fingerprinting?

• Most commonly based on comparisons of repeated DNA sequences between individuals

• Many repeated sequences within our genomes consist of variable copies of the repeated element

• Variable Number Tandem Repeat (VNTR) are typically have repeated elements that are greated than 8 bp. They can be up to a few kb in length

• Short Tandem Repeats (STR or Microsatellites) have repeated units 1-6 bp in length, and are usually less than 200 bp long

What is the method, result, and conclusion of the identification of people based on differences in DNA sequences?

Methods: DNA samples are collected and subjected to PCR; the length of the DNA fragment produced by PCR depends on th4e number of copies of the microsatellite sequence.

Results: The fragments are separated by gel electrophoresis. Different-sized fragments appear as different bands

Conclusion: The patterns of DNA fragments produced by individuals differ

What does the variation in banding patterns reveal?

It reveals inherited differences in microsatellite sequences