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Chapter 2: DNA replication and applications

DNA replication in the cell

Polymerase chain reaction (PCR)

Sequence analysis of DNA

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5’ end: P

3’ end: OH

The importance of 5’ to 3’polarity

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DNA: Summary chemical structure

• DNA consists of two polynucleotide chains which run 5’ to 3’ in opposite directions = antiparallel

• DNA chains are held together by hydrogen bonds between bases

• DNA bases pair by Chargaff’s rules:

- Adenine (A) pairs with Thymine (T)

- Guanine (G) pairs with Cytosine (C)

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DNA Replication

Watson-Crick model of DNA replication

• Hydrogen bonds (weak chemical bond) between DNA bases break to allow strand separation

• Each DNA strand is a template for the synthesis of a new strand

• Template (parental) strand determines the sequence of bases in the new strand (daughter)= complementary base pairing rules

• Enzyme for DNA synthesis = DNA polymerase

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Replication fork:

the two strands of the DNA molecule separate and each serves as a template for DNA synthesis

DNA Replication

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DNA Synthesis: the building blocks

Addition of nucleo-tides into growing DNA chain by DNA polymerase occurs by cleavage of two phosphate groups and the attachment of the nucleoside monophosphate to the 3’-OH of adjacent deoxyribose sugar

DNA Replication

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DNA Replication

• DNA polymerase extends a chain of nucleotides in 5’- to- 3’ direction only, this is the direction of DNA synthesis

• Template strand is 3’-to- 5’, so the complementary DNA strands are antiparallel

• How can both strands be replicated in opposite directions??

Template DNA synthesis

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Intermezzo for the interested persons

Each replication fork consists of:• Leading strand: template is 3’- to-5’and its

synthesis = 5’-to-3’• Lagging strand: template is 3’-to-5’, DNA synthesis

should be 5’- 3’ but its overall direction is 3’-to-5’• DNA polymerase

synthesizes lagging strand in short 5’-to-3’ segments = Okazaki fragments

DNA Replication

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Primers: Role in Replication

• Primer = short RNA segment complementary to DNA at origins of replication synthesized by primase (in vivo definition)

• Primer provides 3’-OH which can be extended by DNA polymerase

• Okazaki fragments are also initiated by primers eventually replaced by DNA

• DNA ligase joins ends

DNA Replication

Intermezzo 2: for those interested

RNA primer is later removed and replaced by DNA

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Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR)

= exponential amplification of DNA in test tube (in vitro)

= used to detect and amplify very small DNA amounts

• DNA sequence to be amplified is targeted using two primers = oligonucleotides = short (ca. 20 nt) synthetic single- stranded DNA segments (in vitro definition).

• A primer binds to the complementary DNA sequence and can be used as starting point for DNA synthesis by DNA polymerase. The two primers for PCR have to bind to the opposite strands pointing towards each other.

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Polymerase Chain Reaction

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• Exponential amplification occurs when this process of primer annealing (hybridisation) and elongation (DNA synthesis) is repeated.

• This repetition of cycles of DNA replication is done by changing the incubation temperatures:1. Denaturation (strand separation): 95 °C2. Primer hybridisation (annealing): variable, determined by the primer length and sequence (40 - 60 °C) (G-C 3 bonds is stronger than A-T 2 bonds, more GC needs a higher temperature to separate the strands)3. Extension (DNA synthesis): 72 °C

Polymerase Chain Reaction

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Polymerase Chain Reaction

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http://vector.cshl.org/resources/BiologyAnimationLibrary.htm

Pcr.exe

Length primer important !

-Too short = non specific

-Longer = more expensive

Polymerase Chain Reaction

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• DNA polymerase used in PCR = Taq polymerase isolated from bacterial thermophiles (Thermus aquaticus) which can withstand high temperature used in procedure

• The primers (made by chemical synthesis) are incorporated in the PCR-product

• The primers are typically around 20 nucleotides long

Polymerase Chain Reaction

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• PCR accomplishes the rapid production of large amounts of target DNA which can then be identified and analyzed

• In normal PCR it is essential to know the outer sequence of the target (to design primers)

• The sensitivity of PCR is a big advantage but can also cause problems (amplification of contaminating DNA on hands or instruments or even from the air)

• The product of a PCR reaction can be analysed by gel electrophoresis: usually one band (or none if the sample is negative for the tested target DNA)

Polymerase Chain Reaction

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DNA fragments can be separated according to

their size by gel electrophoresis: DNA is

negative and moves to the positive pole

•agarose gel electrophoresis (300 bp - 15 kb)

•polyacrylamide gel electrophoresis (1-500 b)

=PAGE

Gelelectrophoresis

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agarose gel electrophoresis (300 bp - 15 kb)

Gelelectrophoresis

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DNA can be detected by staining with ethidiumbromide, that gives an orange fluorescence upon UV- irradiation or by incorporation of radio-active or fluorescent nucleotides

Gelelectrophoresis

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Gelelectrophoresis

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• The sensitivity of PCR: amplification of one molecule of DNA is possible (example: pre-implantation diagnosis)

Polymerase Chain Reaction

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DNA Sequence Analysis

• DNA sequence analysis determines the order of bases in DNA

• Dideoxy method uses DNA bases containing modified deoxyribose sugars = dideoxyribose which contain H at the 3’ position of the ribose sugar rather than OH

• These modified sugars cause chain termination

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Dideoxy Method

• Each of four reactions contains a different dideoxynucleotide = A, T, G, or C in addition to the four bases

• A primer is used to start DNA synthesis

• Synthesis occurs in each reaction tube until a dideoxy base is inserted which results in chain termination

• Each tube contains a set of DNA pieces ending with the same base

DNA Sequence Analysis

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• E.g. test tube with ddGTP results in DNA strands with different lengths, each ending in G (C on the corresponding template)

DNA Sequence Analysis

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Four reactions are set up, each with 1 ddNTP

DNA Sequence Analysis

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• Polyacrylamide gel electrophoresis is used to separate the reaction products from each tube = DNAs end in A, G, T or C

• DNA sequence can be read in the 5’-to-3’ direction from the bottom of gel

• Each band on the gel is one base longer than the previous band

• Bases are identified by gel position

DNA Sequence Analysis

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• Radioactive labeling:

In the original method P32 is incorporated in the synthesised DNA so the results can be visualised on a sensitive film (autoradiography)

DNA Sequence Analysis

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• Fluorescent label • For automatic DNA sequence-analysis

fluorescently labeled ddNTPs are used• The 4 reactions are loaded on 1 lane, the

colour (of the base) is detected after excitation by a laser and the information is directly sent to a computer

DNA Sequence Analysis

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5’

3’

DNA Sequence Analysis

The result!

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3’

5’

Using the table of the genetic code, the protein encoded by the gene can be deduced (done by computer of course)