Chapter 6.5

Question 1

What is meant by recombinant DNA technology

Answer 1

-it describes how a DNA fragment from any source can be multiplied by gene cloning or polymerase chain reaction(PCR)

Question 2

Is DNA to be cloned present in a small quantity? Is it a heterogenous mixture or a homogenous one?

Answer 2

• it is present in small quantities

- >it is a heterogenous mixture to start off with

Question 3

What are vectors? Describe the process of transferring it into a host bacteria.

Answer 3

• they are bacterial or viral plasmids
• > that can be transferred into another bacterium(the host)
• > bacteria are then grown in colonies and a colony containing the recombinant vector is isolated
• > note that the recombinant vector also contains a gene for antibiotic resistance

-note vectors also contain palandromic sequences that can be recognized by restriction enzymes

Question 4

What are restriction enzymes? What kind of strands do they recognize?

Answer 4

• they are enzymes that recognize specific double stranded DNA sequences
• > these sequences are palindromic(meaning that five prime to 3 prime sequence is identical to three prime to five prime strand)

Question 5

Where are restriction enzymes isolated from?

Answer 5

• they are isolated from bacteria

- >bacteria is their natural source

Question 6

What are DNA libraries? How are they made

Answer 6

• large collection of known DNA sequences
• > it could equal to the genome of an organism
• they are made because DNA fragments are often digested randomly
• > are cloned into vectors and utilized for further studies

Question 7

What are genomic libraries

Answer 7

• contain large fragments of DNA
• > include both exon and intron(non coding regions) of DNA

-may contain multiple vectors

Question 8

What are cDNA libraries? How are they made?

Answer 8

• libraries constructed by reverse-transcribing processed mRNA
• > therefore, cDNA lacks noncoding regions such as introns

-note cDna libraries use reverse transcriptase enzymes, while genomic libraries use restriction endonuclease

Question 9

What is meant by the process of hybridization?

Answer 9

• joining of complementary base pair sequences
• this can be DNA-DNA recognition or DNA-RNA recognition

-uses two single stranded sequences

Question 10

How does PCR work? Discuss primers

Answer 10

• they got primers complementary to the DNA
• > primers have high GC content and therefore are stable
• during PCR
• > DNA is denatured
• > replicated
• > and then cooled to allow reannealing of the daughter strands with the parent strands
• > this process is repeated several times, doubling the amount of DNA with each cycle

-the DNA polymerase used for PCR is Thermus aquaticus

Question 11

What kind of gel is used for gel electrophoresis of DNA?

Answer 11

-agarose gel is used

Question 12

What is southern blotting? Describe the process

Answer 12

-it is used to detect the presence and quantity of various DNA strands in a sample

• DNA is cut by restriction enzymes
• > then separated by gel electrophoresis
• > DNA is then transferred to a membrane
• > membrane is then probed with many copies of single-stranded DNA sequence
• probes bind to complementary sequences and form double stranded DNA
• > probes are labelled with radioisotopes or indicator proteins
• > which can be used to indicate the presence of a desired sequence

Question 13

What materials or products are needed for DNA sequencing?

Answer 13

-you need template DNA, primers, an appropriate DNA polymerase, all four doexyribronucleotide triphosphates, and dideoxyribonucleotides in low concentrations

Question 14

What is so special about dideoxyribonucleotides?

Answer 14

• they contain a hydrogen at carbon 3 rather than a hydroxyl group
• > so the polymerase can no longer add to this chain
• eventually, you end up with many fragments
• > each one which terminates with one of the modified dideoxyribonucleotides
• > these fragments are then seperated by size using gel electrophoresis
• > the last base for each fragment is read