BIO- 20

MB: What makes DNA cloning possible?

The ability of restriction enzymes to cut DNA at specific sites

MB: Cloning a gene requires…

…an enzyme that has restriction sites on both sides of that gene but not within the gene

MB: What make it possible for the fragments to combine with the DNA of a cloning vector, such as a plasmid

Sticky ends

MB: To screen a library of bacterial colonies for clones that carry a specific gene…

…a relatively short, single-stranded nucleic acid probe is hybridized to the DNA of that gene

MB: (TRUE / FALSE) The probe will have the same sequence as the DNA template strand (the one you used to make the mRNA)

TRUE

MB: In order to insert a human gene into a plasmid, both must ____________

be cut by the same restriction enzyme, resulting in the formation of complementary sticky ends

MB: What enzyme forms covalent bonds between restriction fragments?

DNA ligase

MB: Using DNA technology to recombine and copy genes

1. Restriction enzyme is used to cut open a plasmid 2. Plasmid serves as a cloning vector

MB: The process by which a bacterium takes up a plasmid from the surrounding solution

Transformation

MB: Occurs with the replication of a recombinant plasmid

Cloning

MB: The unpaired nucleotides produced by the action of restriction enzymes

Sticky ends (they will "stick" to a complementary single-stranded sequence)

MB: Matching sticky ends

The matching of sticky ends follows the rules of specific base pairing

MB: In gene-cloning projects, plasmids function to ________

allow replication of the DNA that’s being cloned

MB: To isolate a cloned gene from a DNA library, researchers ______

probe the library using a labeled single-stranded DNA complementary to the gene

MB: What is the difference between a gene (genomic) library and a gene clone?

A genomic library contains many different DNA sequences; a gene clone contains one type of DNA sequence

MB: If a scientist needs to put new DNA into a bacterium, she must _____ the bacterium

Transform

MB: In recombinant DNA methods, the term vector can refer to _____

A plasmid used to transfer DNA into a living cell

MB: What information can not be obtained from the sequence of a gene?

Whether the gene is methylated

MB: What is the polymerase chain reaction (PCR)?

A method to amplify a fragment of DNA

MB: True or false? Comparison of the sequences of the same gene across species can give some insight into the existence of a common ancestor with that gene.

True

MB: True or false? The Taq enzyme is a type of DNA polymerase that allows researchers to separate the DNA strands during the annealing step of the PCR cycle without destroying the polymerase.

False

MB: How many DNA molecules would there be after four rounds of PCR if the initial reaction mixture contained two molecules?

32

MB: During which step in the PCR cycle are nucleotides used?

Extension

MB: During which step in the PCR cycle do primers form bonds with a single-stranded template?

Annealing

MB: Why is "chain reaction" an appropriate part of the term PCR?

Newly synthesized DNA segments serve as templates in subsequent cycles

MB: What controls which DNA is amplified in PCR?

The choice of primers

MB: Each round of PCR ______

doubles the amount of DNA

MB: What is a primary difference between polymerase chain reaction (PCR) and traditional cloning procedures such as those used to clone the human growth hormone gene?

PCR eliminates the need for restriction enzymes, vectors, and cells

MB: What information is critical to the success of polymerase chain reaction (PCR) itself?

The DNA sequence of the ends of the DNA to be amplified must be known

MB: In a single polymerase chain reaction (PCR) cycle consisting of 15 seconds at 94°C, 30 seconds at 50°C, and 1 minute at 72°C, what is happening in the step run at 50°C?

Primers are annealing to the DNA to be amplified

Genetic Engineering

Removing DNA sequences from an organism, manipulate them, and insert them into different individuals

Role of enzymes in genetic engineering

– cut DNA at specific sites – paste DNA sequences together

Recombinant DNA technology

techniques used to engineer genes

Pituitary gland produces HGH

– stimulates growth – codes for GH1 gene – 191 amino acids

GH1 deficiency

Pituitary dwarfism (slower growth / shorter stature)

Pituitary Dwarfism (Type I)

Autosomal recessive trait (2 copies of defective gene)

Initial treatment of dwarfism

Injections of naturally produced growth hormone

Were natural injections successful?

Only successful when treated with GH from human proteins

Prions

Infectious proteins causing degenerative brain disorders in mammals

Prion Disease

Developed in children treated with HGH therapy

Source of prion disease

Contaminated cadavers

Plan B:

Insert fully functional copies of human GH1 into E. coli to produce huge quantities of recombinant progeny

Reverse transcriptase

Catalyzes the synthesis of DNA from RNA template

Complementary (cDNA)

DNA produced from RNA

Purpose of cDNA

Researchers add a chemically synthesized primer to a single-stranded cDNA and used DNA polymerase to synthesize the second strand

Isolate mRNA’s from…

…pituitary gland cells and use enzymes to reverse-transcribe mRNA→cDNA

Product of reverse transcription

Double-stranded cDNA corresponding to each gene actively expressed

DNA cloning

Producing many copies of a gene

How to clone?

Insert a gene into a small, circular DNA molecule (plasmid)

Replication (passing on plasmids)

Splice loose DNA into a plasmid and insert it into a bacterial cell hoping the bacteria grow/divide

Cloning Vector

Using a plasmid to make copies of a foreign DNA sequence

Restriction Endonuclease

Bacterial enzyme to cut DNA molecules at specific base sequences for later insertion into a cloning vector

Palindrome

Word/Sentence that reads the same way backward as it does forward

DNA palindrome

5’→3′ is identical to the 5’→3′ sequence on the antiparallel, complementary strand

Inserting Genes into Plasmids

1. Identify a palindromic recognition site 2. Add restriction endonuclease 3. Sticky ends result 4. Insert gene into plasmid

1. Identify a palindromic recognition site

– plasmid contains a recognition site for a restriction endonuclease – attach same recognition site to cDNA corresponding to a gene that will be inserted into the plasmid

2. Add restriction enzyme

Makes staggered cuts at each of the recognition sites

3. Sticky ends result

Recognition sites now have "sticky ends" capable of H-bonding with a complimentary sequence

4. Insert Gene into Plasmid

Sticky ends on plasmid and on gene to be inserted bind by complimentary base pairing. DNA ligase catalyzes formation of a phosphodiester bond at points, "sealing" the inserted gene

Recombinant DNA technology

The ability to create novel combinations of DNA sequences by cutting specific sequences and pasting them to new locations

Cloning

If a recombinant can be inserted into a bacterial/yeast cell, foreign DNA will be copied and transmitted to new cells as the host grows/divides

Transformation

Cells that take up DNA from the environment and incorporate it into their genomes

How does one increase plasma membrane permeability?

Chemical treatment/electrical shock

DNA library

– Collection of DNA sequences, each of which is inserted into a vector – provide a way to store DNA fragments from a particular cell type or genome in a form that is accessible for gene cloning

cDNA library

The sequences are cDNAs made from a particular cell type or tissue

Genomic library

The sequences are fragments of DNA from an individual’s genome

DNA probe

single-stranded fragment that will bind to a particular single-stranded complimentary sequence in a mixture of DNA – by binding to the target sequence, the probe marks/distinguishes the fragment containing that sequence

Create a cDNA library

1. Isolate mRNAs (from cells in pituitary gland) 2. Synthesize cDNA from each mRNA using reverse transcriptase 3. Make cDNA double-stranded using reverse transcriptase (or DNA polymerase) 4. Make recombinant plasmid: insert each cDNA in a different plasmid 5. Transformation

5. Transformation

– introduce recombinant plasmids into E. coli cells by making cells permeable to DNA – Each cell contains one type of recombinant plasmid and thus one type of cDNA… collection of cells = cell library

Using a DNA Probe

1. Make probe (single-stranded DNA probe has a label that can be visualized) 2. Expose probe to a collection of single-stranded DNA sequences 3. Find probe… it binds to complementary sequences in target DNA (now labeled and ready to be isolated)

Screening a cDNA library

1. Grow transformed E. coli cells containing plasmids on many plates (each colony = different cDNA) 2. Lay a filter on each plate, then remove… some cells from each colony stick to the filter 3. Treat bacteria with chemicals to break open cells and make DNAs single stranded 4. Probe filters with labeled DNA 5. Find probe binding to the complementary sequence in the cDNA library 6. Identify colony (on original plates, find colony of E. coli cells containing growth hormone gene)

How to use the genetic code to predict possible DNA sequences of GH1?

– deduce a set of possible sequences that could encode the GH1 gene – chemically synthesize set of short, single-stranded DNAs that were complementary to the possible GH1 sequences

Biotechnology

Curing dwarfism with recombinant DNA

The promoter sequence is recognized by…

…RNA polymerase holoenzyme

PCR

Polymerase Chain Reaction… an in vitro DNA synthesis reaction that uses DNA polymerase to replicate a specific section of DNA over and over

Since sequence information is required for PCR…

…start by synthesizing short lengths of single-stranded DNA, matching sequences on either side of a gene

Primers

short sequences

Once primers are bound…

…DNA polymerase extends each new DNA strand 5’→3′

PCR (process)

1. Start with a soln containing template DNA, primers, Taq polymerase, and an abundant supply of the 4 dNTPS 2. Denaturation 3. Primer annealing 4. Extension 5. Repeat 2-4 6. Repeat the cycle 20-30 times

Denaturation

Heating separates strands in double helix

Primer annealing

At lower temps, primers bind to template DNA by complementary base pairing

Extension

Taq polymerase uses dNTPs to synthesize complimentary DNA strand, starting at primer

When steps 2-4 are repeated, what happens?

The copies of DNA double

True or False? Taq polymerase is heat stable

True

Which steps constitute a single PCR cycle?

Denaturation, primer annealing, and extension

A total number of "n" cycles can generate __________ copies

2^n

Neanderthals

So old… most DNA had degraded to tiny fragments

Determining if modern humans shared sequences with Neanderthals

– design primers to bracket a region – produce millions of copies of Neanderthal DNA fragments (sequence entire genome of 3 Neanderthals)

Which if he following lists the steps of creating s human brain cDNA library on the proper order

Isolate mRNAs, synthesize cDNA, Make recombinant plasmid, transform into cells

If mRNAs could be lighted and replicated within plasmids, what enzymes commonly used in recombinant DNA technology would no longer be needed

Reverse transcripase

Which of the following would NOT be true of cDNA produced using human brain tissue as the starting material

It could be modified for use as a probe to detect genes expressed in the brain

Many identical copies of genes clones in bacteria are produced as a result of

Plasmid and bacterial cell replication

In recombinant DNA methods, the term vector can refer to

A plasmid used to transfer DNA into E. Coli

The difference between a gene library and a gene clone is that a gene library…

…contains many different DNA sequences while a gene clone contains one

A gene library from a particular humans retinal cell would be a collection of

Genes that have been sequences from a particular organism

How do we describe transformation in bacteria

Uptake of external DNA into a cell

Which of the following would be most useful for increasing he amount of DNA available for testing

PCR

What is a primary difference between PCR and tradition cloning procedures such as those used to clone the HGH gene

PCR eliminates the need for restriction enzyme, vectors and cells

What information is critical to he success of polymerase chain rxn itself

DNA sequence of the ends of DNA to be amplified must be known

In a single PCR cycle what is happening in the step run at 50•C

Primers are annealing to the DNA

Which of the following is in the correct order for one cycle of PCR

Denature DNA, anneal primers, extend primers

Which of the following dsDNA sequences in is most likely to be recognized as restriction enzyme cutting site

GAATTC CTTAAG