enzyme names to learn 1.reverse transcriptase 2.rna polymerase 3.dna helicase 4.dna ligase 5.dna...

Enzyme names to learn1. Reverse

transcriptase

2. RNA polymerase

3. DNA helicase

4. DNA ligase

5. DNA polymerase

6. Restriction endonuclease

A. Unwinds DNA helix within the molecule

B. Joins nucleotides in DNA replication

C. Joins RNA nucleotides to make mRNA during transcription

D. Obtained from retroviruses. Used to make DNA from mRNA

E. Obtained from bacteria. Cut genes from DNA at specific recognition sequences

F. Joins/splices genes to make recombinant DNA

Enzyme names to learn – correct order

1. Reverse transcriptase

2. RNA polymerase

3. DNA helicase

4. DNA ligase

5. DNA polymerase

6. Restriction endonuclease

A. Obtained from retroviruses. Used to make DNA from mRNA

B. Joins RNA nucleotides to make mRNA during transcription

C. Unwinds DNA helix within the molecule (separates two strands)

D. Joins/splices genes to make recombinant DNA

E. Joins nucleotides in DNA replication

F. Obtained from bacteria. Cut genes from DNA at specific recognition sequences

Syllabus 3.5.8

Genes can be cloned by in vivo and in vitro techniques.

In vivo cloning. The use of restriction endonucleases and ligases to insert DNA fragments into vectors, which are then transferred into host cells.

The identification and growth of transformed host cells to clone the desired DNA fragments.

The importance of “sticky ends”.In vitro cloning. The use of the polymerase chain reaction (PCR) in

cloning DNA fragments.The relative advantages of in vivo and in vitro

cloning

How do we make enough DNA fragments?

Cloning means replicating them identically

What is the difference between cloning in vivo and in vitro?

In vitro gene cloning – the polymerase chain reaction

Aqa p.254-5

Objectives

• What is the polymerase chain reaction?• How does the process work? • Advantages of in vitro and in vivo gene

cloning

Producing multiple copies of DNA

PCR headings

• Introduction (what PCR stands for and what it is for)

• Ingredients needed • Process step 1 (p.254)• Process step 2• Process step 3http://www.maxanim.com/genetics/PCR/PCR.htm

• Advantages of in vitro and

in vivo cloning

http://www.maxanim.com/genetics/PCR/PCR.htm

http://www.maxanim.com/genetics/PCR/PCR.htm

Exemplar work

• See ppt• Next slide

Worksheet PCR

Advantages of in vitro and in vivo gene cloning

• in vitro = PCR• in vivo = using bacteria

In the past, one of the drawbacks in obtaining genetic fingerprintsfrom material present at a crime scene was the very small

quantities of DNA recoverable for analysis

A technique called the polymerase chain reaction was developed in 1983 byKary B. Mullis providing the breakthrough that allowed scientists

to produce multiples copies of a DNA sample within a very short period of time

The polymerase chain reaction (PCR) mimics nature’s way of replicating DNAand is able to generate billions of copies of a DNA sample within a few hours

- the technology allows for cheap and rapid amplification of DNA

The technique involves heating DNA to high temperatures to separate the strands and then using the enzyme DNA polymerase to create new strands

Due to the high temperatures required for the technique, a thermostableDNA polymerase had to be found to avoid the expense of needing to

replenish the enzyme after each round of DNA replication

The Polymerase Chain Reaction (PCR)The Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR)Animation – click below to open

The Polymerase Chain Reaction (PCR)Animation – click below to open

Click me, go on.

The solution to this problem was to use Taq polymerase, derived from Thermusaquaticus, a bacterium that is native to hot springs – this enzyme is able to

withstand the high temperatures (up to 95°C) used in the polymerase chain reaction

C CCTAACA AG G G C CG TATC C CGA C G G AT TTGG T

TC C CGA C G G AT TTGG T

C CCTAACA AG G G C CG TA

The target DNA is first mixed with DNA polymerase and primersand then heated to 95°C to separate the two strands of DNA

The Technique

Primers are short, synthetic DNA fragments that are complementary to the DNA

sequences at either end of theregion of DNA to be copied

TGG T

C CG T


TGG T

C CG T

The Technique

The mixture is now cooled to 55°C to allow the primers to bindto the ends of the separated DNA strands

Polymerase binds to the primers and begins adding basesto form new complementary strands


TGG T

C CG T

T

A

A

G

G

G

C

AT

G

C

C

TC

TA

C

A

G

A

G

C

A

C

The Technique

The mixture is now cooled to 55°C to allow the primers to bindto the ends of the separated DNA strands

Polymerase binds to the primers and begins adding basesto form new complementary strands

C


TGG T T A G C T C T C GG A

T


C CG TAGGAGCAAA CC

Two Identical Copies of the Target DNA SequenceResult From the First Synthesis Cycle

C




C CG TAGGAGCTAAA CCC CCTAACA AG G G TC CGA

C C C CG G GA AT T TTGG T

C CG TCCC T G GG AAAAA


TGG T

C CG T

TGG T

C CG TThe process is now repeated by

first heating the mixture toseparate the strands of the

newly formed DNA molecules

The sample is cooled to allow the primers to attach to the

ends of the DNA strands so thatpolymerase can begin its job ofadding bases to the sequence

C CCTAACA AG G G TC CGA




TGG T

C CG T

TGG T

C CG TAt the end of the second cycle there are four complete DNA molecules

identical to the original target DNA

Cycle 2 Products

The cycle is repeated many times with the number ofDNA molecules doubling with each cycle

This exponential increase creates over a billioncopies of the target DNA within a few hours

Cycle 2 Products

Cycle 3 Products

The number of DNA moleculesdoubles with each cycle


C CCTAACA AG G G TC CGA




TGG T

C CG T

TGG T

C CG T

TGG T

C CG T



Target DNA is heated to separate the strands

When the mixture is cooled, primers bind to the ends of the target strands and polymerase enzymes add bases to complete the complementary strands

C



T


C CG TAGGAGCAAA CCTwo identical DNA molecules are formed

A second cycle is initiated by heating the mixtureonce again to separate the strands of the newly

formed DNA moleculesWhen the mixture is cooled, primers bind to the

ends of the target strands and polymerase enzymes add bases to complete the complementary strands

Four identical copies of the target DNA are formed at the end of the second cycle

This cycle of heating and cooling continuesfor approximately 30 cycles, doubling thenumber of DNA molecules with each cycle

SUMMARY PCR generates billions of copies oftarget DNA within a few hours

enzyme names to learn 1.reverse transcriptase 2.rna polymerase 3.dna helicase 4.dna ligase 5.dna...

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