BISC 200 - Recombinant DNA Technology

Exam 3

Recombinant DNA molecule

two sources of DNA that are put together; a types of DNA sequence that has been constructed in a test tube from two or more distinct DNA sequences (spliced together)

Biotechnology

The use of biological processes, particularly in molecular genetics and recombinant DNA tech

Restriction enzyme

Characteristics - Recognize particular DNA sequences called a restriction site and cuts it out (cleaved); enzymes produced naturally by bacteria; palindromic (read the same in opposite direction)

Example – E. coli

Sticky/cohesive ends – ends of both DNA are spliced using a restriction enzyme and stick together to create a recombinant DNA molecule

What is Gel Electrophoresis and what are some examples of methods used to view fragments that this procedure produces?

A biochemical technique that separates DNA fragments based on size and electrical charge.

EX: autoradiography and ethidium bromide (DNA-binding dye)

What is cloning genes?

identical copies of original piece of DNA are produced

Cloning vector

A stable replicating DNA molecule to which a foreign DNA fragment can be attached

three characteristics of a cloning vector:

•       Origin of replication (ori) will ensure that the vector is replicated within the cell

•       Selectable marker (lacZ gene) enables any cell containing the vector to be selected/identified

•       One or more restriction sites – where the DNA fragment can be inserted

What is transformation when working with cloning vectors?

introduction of plasma vector into bacterial host cell

examples of vectors?

Plasmid, Bacteriophage, BACs, YACs

What is PCR used for?

Quicker method to replicate a particular area within a piece of DNA

amplification

a three step process to replicate a specific area within a piece of DNA quickly

what are the three steps of amplification?

denaturation, annealing, and extension

denaturation

brings heat up to 200 degrees F, and causes DNA molecule to come apart (breaks H-bonds), then cools temp to 100-150 degrees F to promote annealing

annealing

the primers being bound to either side of the piece of DNA that goes through the process of amplification

Then DNA replication process starts; Deoxynucleoside triphosphates are added

extension

add primers

taq polymerase

thermophilic bacterium (likes high temps), extends primers; flank the regions that need to be amplified

polymorphism

presence of two or more variant forms of a DNA sequence that can occur among different individuals or populations

Single Nucleotide polymorphism

(Haplotype) a set of genetic markers located close together on a single chromosome or chromosome region

Restricted Fragment Length Polymorphism

variations in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme

DNA finger printing

VNTRs or SRTs; used to identify genes of an individual

STR

short tandem repeats (more commonly used in DNA profiling)

VNTRs

variable number of Tandem repeats (repeated segments in DNA)

Capillary Electrophoresis

can run multiple DNA samples at one time

Profile probability

numerical probability that someone picked at random from a population will share the same DNA profile as the suspects DNA

Product rule

finding the probability of alleles at multiple loci (tells how many other people can have that same genotypic expression)