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Chapter 16 Recombinant DNA & Genetic Engineering

Essays/Diagrams

Frances Coronel

1. Describe each plasmid in example 1. What effect do restriction enzymes have?

2. What are restriction enzymes and how do they work?

Restriction enzymes are used for genetic engineering. they expose base sequence of a DNA fragment. Enzymes cut phosphate backbones of DNA molecules at specific base sequences called recognition sites. Strands of DNA that have been cut w/ restriction enzymes sometimes have single-stranded tails that readily realign with tails from certain other DNA fragments. This allows removing a specific gene from 1 organism & splicing it

into another. Restriction enzymes originally developed in bacteria as a defense against viruses, who inject DNA

in

bacteria which takes over cell. Bacteria's restriction enzymes cut up viral DNA before it can take over cell.

3.

What are practical applications of restriction enzyme digests?

Restriction enzymes are used to cut DNA & vectors. If one wants to clone a gene/DNA into a vector, one can cut them both with same restriction enzyme & ligate wanted gene into vector. This way, one can clone human gene into plasmid & introduce that plasmid into bacteria where it can produce gene product.

4. Electrophoresis is separation technique. Explain importance of: a. agarose gel, b. positive-negative poles on chamber, c. how does separation of DNA fragments occur?

a. Agarose gel acts as a matrix that slows down DNA segments as they move to opposite charged end of gel. Larger segment will have tougher time moving through gel, while smaller segment will move faster b/c its easier to move it through gel.

b. DNA is slightly negative due to phosphate group on nucleotide so fragments are always attracted to positive

pole. DNA fragments hence migrates towards positive pole because its slightly negative.

c. DNA fragments are neg. charged, & during electrophoresis, they are being pulled to pos. end of machine. But

they all are of different sizes, & won't all travel as fast as each other, that's why bands appear in different places.

5. See diagram.

6. Describe steps of PCR.

PCR amplifies a specific gene from essentially a signal copy of DNA. Essentially, there are THREE steps in the PCR cycle -Denaturation (Separation of DNA strands) is needed when you have double stranded DNA. -Annealing is when you have to attach your primers & polymerase to DNA strand.

-Elongation is when you continually add bases following primers until you fully synthesize a new strand of

DNA.

7. What are practical applications of PCR?

DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; diagnosis of hereditary diseases; identification of genetic fingerprints (used in forensic sciences/paternity testing); and detection and diagnosis of infectious diseases

8. Describe how STRs give each of us a unique DNA fingerprint.

A STR in DNA is a class of polymorphisms that occurs when a pattern of 2 or more nucleotides are repeated &

repeated sequences are directly adjacent to each other. Pattern can range in length from 2 to 10 base pairs & is typically in non-coding intron region, making it junk DNA. By examining several STR loci & counting how

many repeats of a specific STR sequence there are at a given locus, its possible to create a unique genetic profile of an individual.

9. Give 2 applications of cDNA. cDNA libraries are quite useful as they can provide the following:

* Determining complete genome sequence of a given organism (genome project)

* Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering

* Study of function of regulatory sequences in vitro

* Study of genetic mutations in cancer tissues