BIO: 20 – Flashcards

The ability of restriction enzymes to cut DNA at specific sites

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

Sticky ends

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

TRUE

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

DNA ligase

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

Transformation

Cloning

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

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

allow replication of the DNA that's being cloned

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

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

Transform

A plasmid used to transfer DNA into a living cell

Whether the gene is methylated

A method to amplify a fragment of DNA

True

False

32

Extension

Annealing

Newly synthesized DNA segments serve as templates in subsequent cycles

The choice of primers

doubles the amount of DNA

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

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

Primers are annealing to the DNA to be amplified

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

- cut DNA at specific sites - paste DNA sequences together

techniques used to engineer genes

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

Pituitary dwarfism (slower growth / shorter stature)

Autosomal recessive trait (2 copies of defective gene)

Injections of naturally produced growth hormone

Only successful when treated with GH from human proteins

Infectious proteins causing degenerative brain disorders in mammals

Developed in children treated with HGH therapy

Contaminated cadavers

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

Catalyzes the synthesis of DNA from RNA template

DNA produced from RNA

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

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

Double-stranded cDNA corresponding to each gene actively expressed

Producing many copies of a gene

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

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

Using a plasmid to make copies of a foreign DNA sequence

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

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

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

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

- 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

Makes staggered cuts at each of the recognition sites

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

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

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

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

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

Chemical treatment/electrical shock

- 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

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

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

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

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

- 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

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)

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)

- 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

Curing dwarfism with recombinant DNA

...RNA polymerase holoenzyme

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

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

short sequences

...DNA polymerase extends each new DNA strand 5'→3'

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

Heating separates strands in double helix

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

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

The copies of DNA double

True

Denaturation, primer annealing, and extension

2^n

So old... most DNA had degraded to tiny fragments

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

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

Reverse transcripase

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

Plasmid and bacterial cell replication

A plasmid used to transfer DNA into E. Coli

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

Genes that have been sequences from a particular organism

Uptake of external DNA into a cell

PCR

PCR eliminates the need for restriction enzyme, vectors and cells

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

Primers are annealing to the DNA

Denature DNA, anneal primers, extend primers

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