Chapter 15: DNA Technology

genetic engineering

the direct manipulation of an organism’s genes using technology to change its genetic makeup and produce new or improved organisms

recombinant DNA

DNA that has been artificially created by combining genetic material from two or more different sources

nucleic acid probe

a single stranded DNA or RNA molecule that is used to detect a specific complementary sequence of nucleic acid in a sample to locate a specfiic gene

gene cloning

process of producing many identical copies of a gene, where it’s just the gene not the whole individual

restriction enzymes

an enzyme that cuts DNA at a specific sequences creating DNA fragments that can be joined to produce recombinant DNA molecules 

restriction fragments

a DNA fragment produced by cutting a long DNA molecule with a restriction enzyme

cloning vector

DNA molecules which a DNA fragment can be inserted to to form a recombinant DNA molecule for the purpose of cloning

acts as a vehicle for transferring genes from one organism to another and to have it expressed

pBLU

a specific plasmid cloning vector that contains an antibiotic resistance gene an the lacZ gene, making it easy to ID bacterial cells that have taken up the plasmid

nucleic acid hybridization

the process in which complementary strands of DNA or RNA pair with each other through base pairing

denaturation

the process of separating the ds DNA into two ss DNA by disrupting the H-bonds between the complementary base pairs

restriction site

a site with a specific sequence of nucleotides in a DNA molecule that is recognized and cut by a restriction enzyme

complementary DNA (cDNA)

a synthetic DNA that is made from a mRNA template via reverse transcription

sticky ends

single-stranded overhangs produced by REs that allow complementary pairing

DNA ligase

seals gaps in the sugar-phosphate backbone

genomic library

collection of clones containing fragments of an organism’s entire genome

cDNA library

library of cloned expressed genes; contains only exons not the entire genome

multiple cloning site

region in plasmid with many restriction sites

lacZ gene

name of bacterial gene that has been spliced into the pBLU plasmid

the gene that encodes beta galactosidase and used in screening (blue/white colonies)

DNA probe 

a single stranded DNA or RNA sequences that is labeled with a marker to detect the presence of a specific complementary DNA sequence

reverse transcriptase

enzyme that synthesizes DNA from an RNA template

restriction site linker

a short synthetic DNA added to create RE sites at ends of cDNA 

DNA methylation

protects DNA from being cut by restriction enzymes by adding a methyl group

T4 DNA Ligase

a type of ligase used to join DNA fragments

Polymerase chain reaction (PCR) 

a lab technique used to make millions to billions of copies of a specific DNA or DNA sequence

process of DNA amplification that is used to detect tiny amounts of genetic material for a variety of applications like detecting diseases or cancers 

DNA amplification

process of making many copies of a specific DNA sequence

thermocycler

the device used for PCR that heats and cools DNA samples in repeating cycles for denaturation, annealing, and extension

taq polymerase

a heat-stable DNA polymerase enzyme that is crucial to the PCR technique

comes from the bacterium thermus aquaticus

oligonucleotide

a short chain of nucleotides which are the building blocks of DNA and RNA and often used as probes or primers

primers

component in a PCR reaction that locates the gene of interest

RFLPs (restriction fragments length polymorphisms)

variations in DNA sequences that cause different individuals to produce different patterns of restriction fragments

• used in genetic mapping and forensics

gel electrophoresis

a technique that separates DNA, RNA, or proteins by size and charge as they move through a gel under an electric field

polyacrylamide

a type of gel material used for electrophoresis, particularly good for separating very small DNA fragments with high resolution

agarose

a gel that used for separating larger DNA fragments during electrophoresis

DNA fingerprint

a unique pattern of DNA produced by analyzing RFLP, VNTRs, or other markers used to ID in forensics, and paternity cases

VNTR loci (variable number tandem repeats)

regions of DNA where short sequences repeat a variable number of times, differing among individuals and useful in DNA fingerprinting

DNA sequencing

helps determine the exact order of nucleotides in a DNA molecule

dideoxynucleoside triphosphates (ddNTPs)

modified nucleotides used in Sanger sequencing; they stop DNA synthesis when incorporated because they lack the 3’ OH group

Polyacrylamide Gel Electrophoresis

A high-resolution electrophoresis technique used in DNA sequencing to separate small DNA fragments differing by just one nucleotide.

tools used only in the construction of the cDNA library but NOT the genomic library

reverse transcriptase

tools that are used in the construction of the genomic library

1. restriction enzymes

2. DNA ligase

3. plasmid